Bispecific, bivalent anti-vegf/anti-ang2 antibodies

ABSTRACT

The present invention relates to bispecific, bivalent antibodies against human vascular endothelial growth factor (VEGF/VEGF-A) and against human angiopoietin-2 (ANG-2), methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.

RELATED APPLICATION

This application claims priority to and the benefit of EuropeanApplication No. 10003269.7 filed Mar. 26, 2010, the contents of which isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to bispecific, bivalent antibodies againsthuman vascular endothelial growth factor (VEGF/VEGF-A) and against humanangiopoietin-2 (ANG-2), methods for their production, pharmaceuticalcompositions containing said antibodies, and uses thereof.

BACKGROUND OF THE INVENTION

Angiogenesis is implicated in the pathogenesis of a variety of disorderswhich include solid tumors, intraocular neovascular syndromes such asproliferative retinopathies or age-related macular degeneration (AMD),rheumatoid arthritis, and psoriasis (Folkman, J., et al., J. Biol. Chem.267 (1992) 10931-10934; Klagsbrun, M., et al., Annu Rev. Physiol. 53(1991) 217-239; and Garner, A., Vascular diseases, in: Pathobiology ofocular disease, A dynamic approach, Garner, A., and Klintworth, G. K.(eds.), 2nd edition, Marcel Dekker, New York (1994), pp. 1625-1710). Inthe case of solid tumors, the neovascularization allows the tumor cellsto acquire a growth advantage and proliferative autonomy compared to thenormal cells. Accordingly, a correlation has been observed betweendensity of microvessels in tumor sections and patient survival in breastcancer as well as in several other tumors (Weidner, N., et al., N Engl JMed. 324 (1991) 1-8; Horak, E. R., et al., Lancet 340 (1992) 1120-1124;and Macchiarini, P., et al., Lancet 340 (1992) 145-146).

VEGF and anti-VEGF Antibodies

Human vascular endothelial growth factor (VEGF/VEGF-A) (SEQ ID No: 105)is described in e.g. Leung, D. W., et al., Science 246 (1989) 1306-9;Keck, P. J., et al., Science 246 (1989) 1309-12 and Connolly, D. T., etal., J. Biol. Chem. 264 (1989) 20017-24. VEGF is involved in theregulation of normal and abnormal angiogenesis and neovascularizationassociated with tumors and intraocular disorders (Ferrara, N., et al.,Endocr. Rev. 18 (1997) 4-25; Berkman, R. A., et al., J. Clin. Invest. 91(1993) 153-159; Brown, L. F., et al., Human Pathol. 26 (1995) 86-91;Brown, L. F., et al., Cancer Res. 53 (1993) 4727-4735; Mattern, J., etal., Brit. J. Cancer. 73 (1996) 931-934; and Dvorak, H. F., et al., Am.J. Pathol. 146 (1995) 1029-1039). VEGF is a homodimeric glycoproteinthat has been isolated from several sources. VEGF shows highly specificmitogenic activity for endothelial cells. VEGF has important regulatoryfunctions in the formation of new blood vessels during embryonicvasculogenesis and in angiogenesis during adult life (Carmeliet, P., etal., Nature, 380 (1996) 435-439; Ferrara, N., et al., Nature, 380 (1996)439-442; reviewed in Ferrara, N., et al., Endocr. Rev. 18 (1997) 4-25.The significance of the role played by VEGF has been demonstrated instudies showing that inactivation of a single VEGF allele results inembryonic lethality due to failed development of the vasculature(Carmeliet, P., et al., Nature, 380 (1996) 435-439; Ferrara, N., et al.,Nature, 380 (1996) 439-442. In addition VEGF has strong chemoattractantactivity towards monocytes, can induce the plasminogen activator and theplasminogen activator inhibitor in endothelial cells, and can alsoinduce microvascular permeability. Because of the latter activity, it issometimes referred to as vascular permeability factor (VPF). Theisolation and properties of VEGF have been reviewed; see Ferrara, N., etal., J. Cellular Biochem., 47 (1991) 211-218 and Connolly, D. T., J.Cellular Biochem., 47 (1991) 219-223. Alternative mRNA splicing of asingle VEGF gene gives rise to five isoforms of VEGF.

Anti-VEGF neutralizing antibodies suppress the growth of a variety ofhuman tumor cell lines in mice (Kim, K. J., et al., Nature 362 (1993)841-844; Warren, S. R., et al., J. Clin. Invest. 95 (1995) 1789-1797;Borgstrom, P., et al., Cancer Res. 56 (1996) 4032-4039; and Melnyk, O.,et al., Cancer Res. 56 (1996) 921-924). WO 94/10202, WO 98/45332, WO2005/00900 and WO 00/35956 refer to antibodies against VEGF. Humanizedmonoclonal antibody bevacizumab (sold under the trade name Avastin®) isan anti-VEGF antibody used in tumor therapy WO 98/45331).

Ranibizumab (trade name Lucentis®) is a monoclonal antibody fragmentderived from the same parent murine antibody as bevacizumab (Avastin).It is much smaller than the parent molecule and has been affinitymatured to provide stronger binding to VEGF-A (WO 98/45331). It is ananti-angiogenic that has been approved to treat the “wet” type ofage-related macular degeneration (ARMD), a common form of age-relatedvision loss. Another anti-VEGF antibody is e.g. HuMab G6-31 describede.g. in US 2007/0141065.

ANG-2 and Anti-ANG-2 Antibodies

Human angiopoietin-2 (ANG-2) (alternatively abbreviated with ANGPT2 orANG2) (SEQ ID No: 106) is described in Maisonpierre, P. C., et al,Science 277 (1997) 55-60 and Cheung, A. H., et al., Genomics 48 (1998)389-91. The angiopoietins-1 and -2 (ANG-1 (SEQ ID No: 107) and ANG-2(SEQ ID No: 106)) were discovered as ligands for the Ties, a family oftyrosine kinases that is selectively expressed within the vascularendothelium. Yancopoulos, G. D., et al., Nature 407 (2000) 242-48. Thereare now four definitive members of the angiopoietin family.Angiopoietin-3 and -4 (Ang-3 and Ang-4) may represent widely divergedcounterparts of the same gene locus in mouse and man. Kim, I., et al.,FEBS Let, 443 (1999) 353-56; Kim, I., et al., J Biol Chem 274 (1999)26523-28. ANG-1 and ANG-2 were originally identified in tissue cultureexperiments as agonist and antagonist, respectively (see for ANG-1:Davis, S., et al., Cell 87 (1996) 1161-69; and for ANG-2: Maisonpierre,P. C., et al., Science 277 (1997) 55-60) All of the known angiopoietinsbind primarily to Tie2, and both Ang-1 and -2 bind to Tie2 with anaffinity of 3 nM (Kd). Maisonpierre, P. C., et al., Science 277 (1997)55-60. Ang-1 was shown to support EC survival and to promote endotheliumintegrity, Davis, S., et al., Cell 87 (1996) 1161-69; Kwak, H. J., etal., FEBS Lett 448 (1999) 249-53; Suri, C., et al., Science 282 (1998)468-71; Thurston, G., et al., Science 286 (1999) 2511-2514; Thurston,G., et al., Nat. Med. 6 (2000) 460-63, whereas ANG-2 had the oppositeeffect and promoted blood vessel destabilization and regression in theabsence of the survival factors VEGF or basic fibroblast growth factor.Maisonpierre, P. C., et al., Science 277 (1997) 55-60. However, manystudies of ANG-2 function have suggested a more complex situation. ANG-2might be a complex regulator of vascular remodeling that plays a role inboth vessel sprouting and vessel regression. Supporting such roles forANG-2, expression analyses reveal that ANG-2 is rapidly induced,together with VEGF, in adult settings of angiogenic sprouting, whereasANG-2 is induced in the absence of VEGF in settings of vascularregression. Holash, J., et al., Science 284 (1999) 1994-98; Holash, J.,et al., Oncogene 18 (1999) 5356-62. Consistent with a context-dependentrole, ANG-2 specifically binds to the same endothelial-specificreceptor, Tie-2, which is activated by Ang-1, but has context-dependenteffects on its activation. Maisonpierre, P. C., et al., Science 277(1997) 55-60.

Corneal angiogenesis assays have shown that both ANG-1 and ANG-2 hadsimilar effects, acting synergistically with VEGF to promote growth ofnew blood vessels. Asahara, T., et al., Circ. Res. 83 (1998) 233-40. Thepossibility that there was a dose-dependent endothelial response wasraised by the observation that in vitro at high concentration, ANG-2 canalso be pro-angiogenic. Kim, 1., et al., Oncogene 19 (2000) 4549-52. Athigh concentration, ANG-2 acts as an apoptosis survival factor forendothelial cells during serum deprivation apoptosis through activationof Tie2 via PI-3 Kinase and Akt pathway. Kim, I., et al., Oncogene 19(2000) 4549-52.

Other in vitro experiments suggested that during sustained exposure, theeffects of ANG-2 may progressively shift from that of an antagonist toan agonist of Tie2, and at later time points, it may contribute directlyto vascular tube formation and neovessel stabilization.Teichert-Kuliszewska, K., et al., Cardiovasc. Res. 49 (2001) 659-70.Furthermore, if ECs were cultivated on fibrin gel, activation of Tie2with ANG-2 was also observed, perhaps suggesting that the action ofANG-2 could depend on EC differentiation state. Teichert-Kuliszewska,K., et al., Cardiovasc. Res. 49 (2001) 659-70. In microvascular ECcultured in a three-dimensional collagen gel, ANG-2 can also induce Tie2activation and promote formation of capillary-like structures.Mochizuki, Y., et al., J. Cell. Sci. 115 (2002) 175-83. Use of a 3-Dspheroidal coculture as an in-vitro model of vessel maturationdemonstrated that direct contact between ECs and mesenchymal cellsabrogates responsiveness to VEGF, whereas the presence of VEGF and ANG-2induced sprouting. Korff, T., et al., Faseb J. 15 (2001) 447-57. Etoh,T. H. et al. demonstrated that ECs that constitutively express Tie2, theexpression of MMP-1, -9 and u-PA were strongly upregulated by ANG-2 inthe presence of VEGF. Etoh, T., et al., Cancer Res. 61 (2001) 2145-53.With an in vivo pupillary membrane model, Lobov, I. B. et al. showedthat ANG-2 in the presence of endogenous VEGF promotes a rapid increasein capillary diameter, remodeling of the basal lamina, proliferation andmigration of endothelial cells, and stimulates sprouting of new bloodvessels. Lobov, I. B., et al., Proc. Natl. Acad. Sci. USA 99 (2002)11205-10. By contrast, ANG-2 promotes endothelial cell death and vesselregression without endogenous VEGF. Lobov, I. B., et al., Proc. Natl.Acad. Sci. USA 99 (2002) 11205-10. Similarly, with an in vivo tumormodel, Vajkoczy, P., et al. demonstrated that multicellular aggregatesinitiate vascular growth by angiogenic sprouting via the simultaneousexpression of VEGFR-2 and ANG-2 by host and tumor endothelium. Vajkoczy,P., et al., J. Clin. Invest. 109 (2002) 777-85. This model illustratedthat the established microvasculature of growing tumors is characterizedby a continuous remodeling, putatively mediated by the expression ofVEGF and ANG-2 (Vajkoczy, P., et al., J. Clin. Invest. 109 (2002)777-85).

Knock-out mouse studies of Tie-2 and Angiopoietin-1 show similarphenotypes and suggest that Angiopoietin-1 stimulated Tie-2phosphorylation mediates remodeling and stabilization of developingvessel, promoting blood vessel maturation during angiogenesis andmaintenance of endothelial cell-support cell adhesion (Dumont, D. J., etal., Genes & Development, 8 (1994) 1897-1909; Sato, T. N., Nature, 376(1995) 70-74; (Thurston, G., et al., Nature Medicine 6 (2000) 460-463).The role of Angiopoietin-1 is thought to be conserved in the adult,where it is expressed widely and constitutively (Hanahan, D., Science,277 (1997) 48-50; Zagzag, D., et al., Exp Neurology 159 (1999) 391-400).In contrast, Angiopoietin-2 expression is primarily limited to sites ofvascular remodeling where it is thought to block the constitutivestabilizing or maturing function of Angiopoietin-1, allowing vessels torevert to, and remain in, a plastic state which may be more responsiveto sprouting signals (Hanahan, D., 1997; Holash, J., et al., Oncogene 18(199) 5356-62; Maisonpierre, P. C., 1997). Studies of Angiopoietin-2expression in pathological angiogenesis have found many tumor types toshow vascular Angiopoietin-2 expression (Maisonpierre, P. C., et al.,Science 277 (1997) 55-60). Functional studies suggest Angiopoietin-2 isinvolved in tumor angiogenesis and associate Angiopoietin-2overexpression with increased tumor growth in a mouse xenograft model(Ahmad, S. A., et al., Cancer Res., 61 (2001) 1255-1259). Other studieshave associated Angiopoictin-2 overexpression with tumorhypervascularity (Etoh, T., et al., Cancer Res. 61 (2001) 2145-53;Tanaka, F., et al., Cancer Res. 62 (2002) 7124-7129).

In recent years Angiopoietin-1, Angiopoietin-2 and/or Tie-2 have beenproposed as possible anti-cancer therapeutic targets. For example U.S.Pat. No. 6,166,185, U.S. Pat. No. 5,650,490 and U.S. Pat. No. 5,814,464each disclose anti-Tie-2 ligand and receptor antibodies. Studies usingsoluble Tie-2 were reported to decrease the number and size of tumors inrodents (Lin, 1997; Lin 1998). Siemeister, G., et al., Cancer Res. 59:3(1999) 3185-91 generated human melanoma cell lines expressing theextracellular domain of Tie-2, injected these into nude mice andreported soluble Tie-2 to result in significant inhibition of tumorgrowth and tumor angiogenesis. Given both Angiopoietin-1 andAngiopoietin-2 bind to Tie-2, it is unclear from these studies whetherAngiopoietin-1, Angiopoietin-2 or Tie-2 would be an attractive targetfor anti-cancer therapy. However, effective anti-Angiopoietin-2 therapyis thought to be of benefit in treating diseases such as cancer, inwhich progression is dependant on aberrant angiogenesis where blockingthe process can lead to prevention of disease advancement (Folkman, J.,Nature Medicine. 1 (1995) 27-31).

In addition some groups have reported the use of antibodies and peptidesthat bind to Angiopoietin-2. See, for example, U.S. Pat. No. 6,166,185and US 2003/10124129. WO 03/030833, WO 2006/068953, WO 03/057134 or US2006/0122370.

Study of the effect of focal expression of Angiopoietin-2 has shown thatantagonizing the Angiopoietin-1/Tie-2 signal loosens the tight vascularstructure thereby exposing ECs to activating signals from angiogenesisinducers, e.g. VEGF (Hanahan, D., Science, 277 (1997) 48-50). Thispro-angiogenic effect resulting from inhibition of Angiopoietin-1indicates that anti-Angiopoietin-1 therapy would not be an effectiveanti-cancer treatment.

ANG-2 is expressed during development at sites where blood vesselremodeling is occurring. Maisonpierre, P. C., et al., Science 277 (1997)55-60. In adult individuals, ANG-2 expression is restricted to sites ofvascular remodeling as well as in highly vascularized tumors, includingglioma, Osada, H., et al., Int. J. Oncol. 18 (2001) 305-09); Koga, K.,et al., Cancer Res. 61 (2001) 6248-54, hepatocellular carcinoma, Tanaka,S., et al., J. Clin. Invest. 103 (1999) 341-45, gastric carcinoma, Etoh,T., et al., Cancer Res. 61 (2001) 2145-53; Lee, J. H., et al., Int. J.Oncol. 18 (2001) 355-61, thyroid tumor, Bunone, G., et al., Am J Pathol155 (1999) 1967-76 non-small cell lung cancer, Wong, M. P., et al., LungCancer 29 (2000) 11-22, and cancer of colon, Ahmad, S. A., et al.,Cancer 92 (2001) 1138-43, and prostate Wurmbach, J. H., et al.,Anticancer Res. 20 (2000) 5217-20. Some tumor cells are found to expressANG-2. For example, Tanaka, S., et al., J. Clin. Invest. 103 (1999)341-45 detected ANG-2 mRNA in 10 out of 12 specimens of humanhepatocellular carcinoma (HCC). Ellis' group reported that ANG-2 isexpressed ubiquitously in tumor epithelium. Ahmad, S. A., et al., Cancer92 (2001) 1138-43. Other investigators reported similar findings. Chen,L., et al., J. Tongji Med. Univ. 21 (2001) 228-35. By detecting ANG-2mRNA levels in archived human breast cancer specimens, Sfiligoi, C., etal., Int. J. Cancer 103 (2003) 466-74 reported that ANG-2 mRNA issignificantly associated with auxiliary lymph node invasion, shortdisease-free time and poor overall survival. Tanaka, F., et al., CancerRes. 62 (2002) 7124-29 reviewed a total of 236 patients of non-smallcell lung cancer (NSCLC) with pathological stage-I to -IIIA,respectively. Using immunohistochemistry, they found that 16.9% of theNSCLC patients were ANG-2 positive. The microvessel density for ANG-2positive tumor is significantly higher than that of ANG-2 negative. Suchan angiogenic effect of ANG-2 was seen only when VEGF expression washigh. Moreover, positive expression of ANG-2 was a significant factor topredict a poor postoperative survival. Tanaka, F., et al., Cancer Res.62 (2002) 7124-7129. However, they found no significant correlationbetween Ang-1 expression and the microvessel density. Tanaka, F., etal., Cancer Res. 62 (2002) 7124-7129. These results suggest that ANG-2is an indicator of poor prognosis patients with several types of cancer.

Recently, using an ANG-2 knockout mouse model, Yancopoulos' groupreported that ANG-2 is required for postnatal angiogenesis. Gale, N. W.,et al., Dev. Cell 3 (2002) 411-23. They showed that the developmentallyprogrammed regression of the hyaloid vasculature in the eye does notoccur in the ANG-2 knockout mice and their retinal blood vessels fail tosprout out from the central retinal artery. Gale, N. W., et al., Dev.Cell 3 (2002) 411-23. They also found that deletion of ANG-2 results inprofound defects in the patterning and function of the lymphaticvasculature. Gale, N. W., et al., Dev. Cell 3 (2002) 411-23. Geneticrescue with Ang-1 corrects the lymphatic, but not the angiogenesisdefects. Gale, N. W., et al., Dev. Cell 3 (2002) 411-23.

Peters and his colleagues reported that soluble Tie2, when deliveredeither as recombinant protein or in a viral expression vector, inhibitedin vivo growth of murine mammary carcinoma and melanoma in mouse models.Lin, P., et al., Proc. Natl. Acad. Sci. USA 95 (1998) 8829-34; Lin, P.,et al., J. Clin. Invest. 100 (1997) 2072-78. Vascular densities in thetumor tissues so treated were greatly reduced. In addition, soluble Tie2blocked angiogenesis in the rat corneal stimulated by tumor cellconditioned media. Lin, P., et al., J. Clin. Invest. 100 (1997) 2072-78.Furthermore, Isner and his team demonstrated that addition of ANG-2 toVEGF promoted significantly longer and more circumferentialneovascularity than VEGF alone. Asahara, T., et al., Circ. Res. 83(1998) 233-40. Excess soluble Tie2 receptor precluded modulation ofVEGF-induced neovascularization by ANG-2. Asahara, T., et al., Circ.Res. 83 (1998) 233-40. Siemeister, G., et al., Cancer Res. 59:3 (1999)3185-91 showed with nude mouse xenografts that overexpression of theextracellular ligand-binding domains of either Flt-1 or Tie2 in thexenografts results in significant inhibition of pathway could not becompensated by the other one, suggesting that the VEGF receptor pathwayand the Tie2 pathway should be considered as two independent mediatorsessential for the process of in vivo angiogenesis. Siemeister, G., etal., Cancer Res. 59:3 (1999) 3185-91. This is proven by a more recentpublication by White, R., R., et al., Proc. Natl. Acad. Sci. USA 100(2003) 5028-33. In their study, it was demonstrated that anuclease-resistant RNA aptamer that specifically binds and inhibitsANG-2 significantly inhibited neovascularization induced by bFGF in therat corneal micropocket angiogenesis model.

Bispecific Antibodies

A wide variety of recombinant antibody formats have been developed inthe recent past, e.g. tetravalent bispecific antibodies by fusion of,e.g., an IgG antibody format and single chain domains (see e.g. Coloma,M. J., et al., Nature Biotech 15 (1997) 159-163; WO 2001/077342; andMorrison, S. L., Nature Biotech 25 (2007) 1233-1234).

Also several other new formats wherein the antibody core structure (IgA,IgD, IgE, IgG or IgM) is no longer retained such as dia-, tria- ortetrabodies, minibodies, several single chain formats (scFv, Bis-scFv),which are capable of binding two or more antigens, have been developed(Holliger, P., et al., Nature Biotech 23 (2005) 1126-1136; Fischer, N.,Leger, O., Pathobiology 74 (2007) 3-14; Shen, J., et al., Journal ofImmunological Methods 318 (2007) 65-74; Wu, C., et al., Nature Biotech.25 (2007) 1290-1297).

All such formats use linkers either to fuse the antibody core (IgA, IgD,IgE, IgG or IgM) to a further binding protein (e.g. scFv) or to fusee.g. two Fab fragments or scFvs (Fischer, N., Léger, O., Pathobiology 74(2007) 3-14). It has to be kept in mind that one may want to retaineffector functions, such as e.g. complement-dependent cytotoxicity (CDC)or antibody dependent cellular cytotoxicity (ADCC), which are mediatedthrough the Fc receptor binding, by maintaining a high degree ofsimilarity to naturally occurring antibodies.

In WO 2007/024715 are reported dual variable domain immunoglobulins asengineered multivalent and multispecific binding proteins. A process forthe preparation of biologically active antibody dimers is reported inU.S. Pat. No. 6,897,044. Multivalent F_(V) antibody construct having atleast four variable domains which are linked with each over via peptidelinkers are reported in U.S. Pat. No. 7,129,330. Dimeric and multimericantigen binding structures are reported in US 2005/0079170. Tri- ortetra-valent monospecific antigen-binding protein comprising three orfour Fab fragments bound to each other covalently by a connectingstructure, which protein is not a natural immunoglobulin are reported inU.S. Pat. No. 6,511,663. In WO 2006/020258 tetravalent bispecificantibodies are reported that can be efficiently expressed in prokaryoticand eukaryotic cells, and are useful in therapeutic and diagnosticmethods. A method of separating or preferentially synthesizing dimerswhich are linked via at least one interchain disulfide linkage fromdimers which are not linked via at least one interchain disulfidelinkage from a mixture comprising the two types of polypeptide dimers isreported in US 2005/0163782. Bispecific tetravalent receptors arereported in U.S. Pat. No. 5,959,083. Engineered antibodies with three ormore functional antigen binding sites are reported in WO 2001/077342.

Multispecific and multivalent antigen-binding polypeptides are reportedin WO 1997/001580. WO 1992/004053 reports homoconjugates, typicallyprepared from monoclonal antibodies of the IgG class which bind to thesame antigenic determinant are covalently linked by syntheticcross-linking. Oligomeric monoclonal antibodies with high avidity forantigen are reported in WO 1991/06305 whereby the oligomers, typicallyof the IgG class, are secreted having two or more immunoglobulinmonomers associated together to form tetravalent or hexavalent IgGmolecules. Sheep-derived antibodies and engineered antibody constructsare reported in U.S. Pat. No. 6,350,860, which can be used to treatdiseases wherein interferon gamma activity is pathogenic. In US2005/0100543 are reported targetable constructs that are multivalentcarriers of bi-specific antibodies, i.e., each molecule of a targetableconstruct can serve as a carrier of two or more bi-specific antibodies.Genetically engineered bispecific tetravalent antibodies are reported inWO 1995/009917. In WO 2007/109254 stabilized binding molecules thatconsist of or comprise a stabilized scFv are reported.

Combination of VEGF and ANG-2 Inhibitors

WO 2007/068895 refers to a combination of an ANG-2 antagonist and aVEGF, KDR and/or FLTL antagonists. WO 2007/089445 refers to ANG-2 andVEGF inhibitor combinations.

WO 2003/106501 refers to fusion proteins binding to Angiopoietin andcontaining a multimerization domain. WO 2008/132568 relates to fusionproteins binding to Angiopoietin and VEGF. WO 2003/020906 relates tomultivalent protein conjugates with multiple ligand-binding domains ofreceptors.

WO 2009/136352 relates to anti-angiogenic compounds.

SUMMARY OF THE INVENTION

The invention is directed to a bispecific, bivalent antibody comprisinga first antigen-binding site that specifically binds to human VEGF and asecond antigen-binding site that specifically binds to human ANG-2,characterized in that

-   -   i) said first antigen-binding site comprises as heavy chain        variable domain (VH) the SEQ ID NO: 1, and as light chain        variable domain (VL) the SEQ ID NO: 2; and    -   ii) said second antigen-binding site comprises as heavy chain        variable domain (VH) the SEQ ID NO: 3, and as light chain        variable domain (VL) the SEQ ID NO: 4.

In one aspect of the invention the bispecific antibody according to theinvention is characterized in comprising

-   -   a) the heavy chain and the light chain of a first full length        antibody that specifically binds to VEGF; and    -   b) the modified heavy chain and modified light chain of a full        length antibody that specifically binds to ANG-2, wherein the        constant domains CL and CH1 are replaced by each other.

In one embodiment such bispecific, bivalent antibody is characterized incomprising

-   -   a) as heavy chain of the first full length antibody the amino        acid sequence of SEQ ID NO: 7, and as light chain of the first        full length antibody the amino acid sequence of SEQ ID NO: 5,        and    -   b) as modified heavy chain of the second full length antibody        the amino acid sequence of SEQ ID NO: 8, and as modified light        chain of the second full length antibody the amino acid sequence        of SEQ ID NO: 6.

In one embodiment such bispecific, bivalent antibody is characterized incomprising

-   -   a) as heavy chain of the first full length antibody the amino        acid sequence of SEQ ID NO: 11, and as light chain of the first        full length antibody the amino acid sequence of SEQ ID NO: 9,        and    -   b) as modified heavy chain of the second full length antibody        the amino acid sequence of SEQ ID NO: 12, and as modified light        chain of the second full length antibody the amino acid sequence        of SEQ ID NO: 10.

In one embodiment such bispecific, bivalent antibody is characterized incomprising

-   -   a) as heavy chain of the first full length antibody the amino        acid sequence of SEQ ID NO: 15, and as light chain of the first        full length antibody the amino acid sequence of SEQ ID NO: 13,        and    -   b) as modified heavy chain of the second full length antibody        the amino acid sequence of SEQ ID NO: 16, and as modified light        chain of the second full length antibody the amino acid sequence        of SEQ ID NO: 14.

Still further aspects of the invention are a pharmaceutical compositioncomprising said bispecific antibody, said composition for the treatmentof cancer, the use of said bispecific antibody for the manufacture of amedicament for the treatment of cancer, a method of treatment of patientsuffering from cancer by administering said bispecific antibody. to apatient in the need of such treatment.

Still further aspects of the invention are a pharmaceutical compositioncomprising said bispecific antibody, said composition for the treatmentof vascular diseases, the use of said bispecific antibody for themanufacture of a medicament for the treatment of vascular diseases, amethod of treatment of patient suffering from vascular diseases byadministering said bispecific antibody. to a patient in the need of suchtreatment.

A further aspect of the invention is a nucleic acid molecule encoding achain of a bispecific antibody according to the invention.

The invention further provides expression vectors containing saidnucleic acid according to the invention capable of expressing saidnucleic acid in a prokaryotic or eukaryotic host cell, and host cellscontaining such vectors for the recombinant production of a bispecificantibody according to the invention.

The invention further comprises a prokaryotic or eukaryotic host cellcomprising a vector according to the invention.

The invention further comprises a method for the production of abispecific antibody according to the invention, characterized byexpressing a nucleic acid according to the invention in a prokaryotic oreukaryotic host cell and recovering said bispecific antibody from saidcell or the cell culture supernatant. The invention further comprisesthe antibody obtained by such method for the production of a bispecificantibody.

Accordingly one embodiment of the invention is a bispecific, bivalentantibody comprising a first antigen-binding site that specifically bindsto human VEGF and a second antigen-binding site that specifically bindsto human ANG-2, characterized in comprising the amino acid sequences ofSEQ ID NO: 5, of SEQ ID NO: 6, of SEQ ID NO: 7, and of SEQ ID NO: 8.

Accordingly one embodiment of the invention is a bispecific, bivalentantibody comprising a first antigen-binding site that specifically bindsto human VEGF and a second antigen-binding site that specifically bindsto human ANG-2, characterized in comprising the amino acid sequences ofSEQ ID NO: 9, of SEQ ID NO: 10, of SEQ ID NO: 11, and of SEQ ID NO: 12.

Accordingly one embodiment of the invention is a bispecific, bivalentantibody comprising a first antigen-binding site that specifically bindsto human VEGF and a second antigen-binding site that specifically bindsto human ANG-2, characterized in comprising the amino acid sequences ofSEQ ID NO: 13, of SEQ ID NO: 14, of SEQ ID NO: 15, and of SEQ ID NO: 16.

The bispecific, bivalent antibodies according to the invention showbenefits for human patients in need of a VEGF and ANG-2 targetingtherapy. The antibodies according to the invention have highly valuableproperties causing a benefit for a patient suffering from such adisease, especially suffering from cancer. The bispecific antibodiesaccording to the invention are highly effective in tumor growth and/orinhibition of tumor angiogenesis or vascular diseases. The bispecific,bivalent antibodies according to the invention The bispecific, bivalent<VEGF-ANG-2> antibodies according to the invention show valuablepharmacokinetic/-dynamic properties like e.g. stability, good (i.e.slow) clearance (e.g. at low doses).

The bispecific antibodies according to the invention are highlyeffective in

a) tumor growth inhibition (e.g. with the bispecific antibodiesaccording to the invention tumor stasis could be achieved already atlower concentrations compared to the combination of the two monospecificantibodies (e.g. in the Colo205 and the KPL-4 tumor models of Example 9and 10, tumor stasis was already achieved with 10 mg/kg XMAb1 comparedto the combination of 10 mg/kg of ANG2i-LC06+10 mg/kg of Avastin),and/orb) inhibition of tumor angiogenesis or vascular diseases (e.g. maximalantiangiogenic effects with the bispecific antibodies according to theinvention could already be achieved at lower concentrations compared tothe combination of the two monospecific antibodies (e.g. in the mousecorneal angiogenesis assay of Example 8, the maximal antiangiogeniceffect was already achieved with 10 mg/kg XMAb1 compared to thecombination of 10 mg/kg of ANG2i-LC06+10 mg/kg of Avastin).

DESCRIPTION OF THE FIGURES

FIG. 1 Exemplary bivalent bispecific antibody format for XMab examplesincluding Knobs-into-Holes modified CH3 domains

FIG. 2 a Exemplary bivalent bispecific antibody format for OAscFabexamples including Knobs-into-Holes modified CH3 domains

FIG. 2 b Exemplary bivalent bispecific antibody format for exampleOAscXFab1 including Knobs-into-Holes modified CH3 domains

FIG. 2 c Exemplary bivalent bispecific antibody format for examplesOAscXFab2 and OAscXFab3 including Knobs-into-Holes modified CH3 domains

FIG. 3 Simultaneous binding of <VEGF-Ang-2> XMab1 to VEGF (1. Step)followed by binding to hAng-2 (second step)

FIG. 4 ELISA principle for quantification of binding activemAb<Ang2/VEGF> antibodies

FIG. 5 Calibration curve of ELISA for quantification of binding active<Ang2/VEGF> XMab1 antibodies

FIG. 6 Mouse corneal angiogenesis assay—inhibition of vessel outgrowthfrom the limbus towards the VEGF gradient by administration of abispecific antibody according to the invention.

FIG. 7 Mouse corneal angiogenesis assay—inhibition ofangiogenesis/vessel outgrowth from the limbus towards the VEGF gradientby administration of a bispecific antibody according to theinvention—Comparison of the bispecific <Ang2/VEGF> antibody XMab1, the<Ang2> Mab ANG2i-LC06 (LC06), the <VEGF> Mab bevacizumab (Avastin) andthe combination ANG2i-LC06 and <VEGF> Mab bevacizumab (Avastin).

FIG. 8 In vivo tumor growth inhibition in mouse xenograft of humancolorectal cancer Colo205 (small tumors) by a bispecific antibodyaccording to the invention—Comparison of the bispecific <Ang2/VEGF>antibody XMab1, the <Ang2> Mab ANG2i-LCO6 (LC06), the <VEGF> Mabbevacizumab (Avastin) and the combination ANG2i-LC06 and <VEGF> Mabbevacizumab (Avastin).

FIG. 9 In vivo tumor growth inhibition in mouse xenograft of humancolorectal cancer Colo205 (large tumors) by a bispecific antibodyaccording to the invention—Comparison of the bispecific <Ang2/VEGF>antibody XMab1, the <Ang2> Mab ANG2i-LC06 (LC06), the <VEGF> Mabbevacizumab (Avastin) and the combination ANG2i-LC06 and <VEGF> Mabbevacizumab (Avastin).

FIG. 10 In vivo tumor growth inhibition in mouse xenograft of humanbreast cancer KPL-4 (small tumors) by a bispecific antibody according tothe invention—Comparison of the bispecific <Ang2/VEGF> antibody XMab1,the <Ang2> Mab ANG2i-LCO6 (LC06), the <VEGF> Mab bevacizumab (Avastin)and the combination ANG2i-LC06 and <VEGF> Mab bevacizumab (Avastin).

FIG. 11 In vivo tumor growth inhibition in mouse xenograft of humanbreast cancer KPL-4 (large tumors) by a bispecific antibody according tothe invention—Comparison of the bispecific <Ang2/VEGF> antibody XMab1,the <Ang2> Mab ANG2i-LCO6 (LC06), the <VEGF> Mab bevacizumab (Avastin)and the combination ANG2i-LC06 and <VEGF> Mab bevacizumab (Avastin).

FIG. 12 In vivo tumor growth inhibition in mouse xenograft of gastriccancer N87 by a bispecific antibody according to theinvention—Comparison of the bispecific <Ang2/VEGF> antibody XMab1, the<Ang2> Mab ANG2i-LC06 (LC06), the <VEGF> Mab bevacizumab (Avastin) andthe combination ANG2i-LC06 and <VEGF> Mab bevacizumab (Avastin).

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to a bispecific, bivalent antibody comprisinga first antigen-binding site that specifically binds to human VEGF and asecond antigen-binding site that specifically binds to human ANG-2.characterized in that

-   -   i) said first antigen-binding site comprises as heavy chain        variable domain (VH) the SEQ ID NO: 1, and as light chain        variable domain (VL) the SEQ ID NO: 2; and    -   ii) said second antigen-binding site comprises as heavy chain        variable domain (VH) the SEQ ID NO: 3, and as light chain        variable domain (VL) the SEQ ID NO: 4.

In one aspect of the invention the bispecific antibody according to theinvention is characterized in comprising

-   -   a) the heavy chain and the light chain of a first full length        antibody that specifically binds to VEGF;

b) the modified heavy chain and modified light chain of a full lengthantibody that specifically binds to ANG-2, wherein the constant domainsCL and CH1 are replaced by each other.

This bispecific, bivalent antibody format for the bispecific antibodyspecifically binding to human vascular endothelial growth factor (VEGF)and human angiopoietin-2 (ANG-2) is described in WO 2009/080253 (seeexemplary scheme in including Knobs-into-Holes modified CH3 domains inFIG. 1). The antibodies based on this bispecific, bivalent antibodyformat are named XMab in the examples of the current invention.

In one embodiment such bispecific, bivalent antibody is characterized incomprising

-   -   a) as heavy chain of the first full length antibody the amino        acid sequence of SEQ ID NO: 7, and as light chain of the first        full length antibody the amino acid sequence of SEQ ID NO: 5,        and    -   b) as modified heavy chain of the second full length antibody        the amino acid sequence of SEQ ID NO: 8, and as modified light        chain of the second full length antibody the amino acid sequence        of SEQ ID NO: 6.

In one embodiment such bispecific, bivalent antibody is characterized incomprising

-   -   a) as heavy chain of the first full length antibody the amino        acid sequence of SEQ ID NO: 11, and as light chain of the first        full length antibody the amino acid sequence of SEQ ID NO: 9,        and    -   b) as modified heavy chain of the second full length antibody        the amino acid sequence of SEQ ID NO: 12, and as modified light        chain of the second full length antibody the amino acid sequence        of SEQ ID NO: 10.

In one embodiment such bispecific, bivalent antibody is characterized incomprising

-   -   a) as heavy chain of the first full length antibody the amino        acid sequence of SEQ ID NO: 15, and as light chain of the first        full length antibody the amino acid sequence of SEQ ID NO: 13,        and    -   b) as modified heavy chain of the second full length antibody        the amino acid sequence of SEQ ID NO: 16, and as modified light        chain of the second full length antibody the amino acid sequence        of SEQ ID NO: 14.

In one embodiment such bispecific, bivalent antibody is characterized incomprising

-   -   a) as heavy chain of the first full length antibody the amino        acid sequence of SEQ ID NO: 19, and as light chain of the first        full length antibody the amino acid sequence of SEQ ID NO: 17,        and    -   b) as modified heavy chain of the second full length antibody        the amino acid sequence of SEQ ID NO: 20, and as modified light        chain of the second full length antibody the amino acid sequence        of SEQ ID NO: 18.

In one embodiment such bispecific, bivalent antibody is characterized incomprising

-   -   a) as heavy chain of the first full length antibody the amino        acid sequence of SEQ ID NO: 23, and as light chain of the first        full length antibody the amino acid sequence of SEQ ID NO: 21,        and    -   b) as modified heavy chain of the second full length antibody        the amino acid sequence of SEQ ID NO: 24, and as modified light        chain of the second full length antibody the amino acid sequence        of SEQ ID NO: 22.

In one embodiment such bispecific, bivalent antibody is characterized incomprising

-   -   a) as heavy chain of the first full length antibody the amino        acid sequence of SEQ ID NO: 27, and as light chain of the first        full length antibody the amino acid sequence of SEQ ID NO: 25,        and    -   b) as modified heavy chain of the second full length antibody        the amino acid sequence of SEQ ID NO: 28, and as modified light        chain of the second full length antibody the amino acid sequence        of SEQ ID NO: 26.

Accordingly one embodiment of the invention is a bispecific, bivalentantibody comprising a first antigen-binding site that specifically bindsto human VEGF and a second antigen-binding site that specifically bindsto human ANG-2, characterized in comprising the amino acid sequences ofSEQ ID NO: 5, of SEQ ID NO: 6, of SEQ ID NO: 7, and of SEQ ID NO: 8.

Accordingly one embodiment of the invention is a bispecific, bivalentantibody comprising a first antigen-binding site that specifically bindsto human VEGF and a second antigen-binding site that specifically bindsto human ANG-2, characterized in comprising the amino acid sequences ofSEQ ID NO: 9, of SEQ ID NO: 10, of SEQ ID NO: 11, and of SEQ ID NO: 12.

Accordingly one embodiment of the invention is a bispecific, bivalentantibody comprising a first antigen-binding site that specifically bindsto human VEGF and a second antigen-binding site that specifically bindsto human ANG-2, characterized in comprising the amino acid sequences ofSEQ ID NO: 13, of SEQ ID NO: 14, of SEQ ID NO: 15, and of SEQ ID NO: 16.

Accordingly one embodiment of the invention is a bispecific, bivalentantibody comprising a first antigen-binding site that specifically bindsto human VEGF and a second antigen-binding site that specifically bindsto human ANG-2, characterized in comprising the amino acid sequences ofSEQ ID NO: 17, of SEQ ID NO: 18, of SEQ ID NO: 19, and of SEQ ID NO: 20.

Accordingly one embodiment of the invention is a bispecific, bivalentantibody comprising a first antigen-binding site that specifically bindsto human VEGF and a second antigen-binding site that specifically bindsto human ANG-2, characterized in comprising the amino acid sequences ofSEQ ID NO: 21, of SEQ ID NO: 22, of SEQ ID NO: 23, and of SEQ ID NO: 24.

Accordingly one embodiment of the invention is a bispecific, bivalentantibody comprising a first antigen-binding site that specifically bindsto human VEGF and a second antigen-binding site that specifically bindsto human ANG-2, characterized in comprising the amino acid sequences ofSEQ ID NO: 25, of SEQ ID NO: 26, of SEQ ID NO: 27, and of SEQ ID NO: 28.

In another aspect of the invention the bispecific antibody according tothe invention is characterized in comprising

-   -   a) the heavy chain and the light chain of a first full length        antibody that specifically binds to VEGF;    -   b) the heavy chain and the light chain of a second full length        antibody that specifically binds to ANG-2, wherein the        N-terminus of the heavy chain is connected to the C-terminus of        the light chain via a peptide linker.

An exemplary scheme of this bispecific, bivalent antibody format forthis bispecific antibody specifically binding to human vascularendothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) isshown in FIG. 2 a including Knobs-into-Holes modified CH3 domains. Theantibodies based on this bispecific, bivalent antibody format are namedOAscFab in the examples of the current invention.

In one embodiment such bispecific, bivalent antibody is characterized incomprising

-   -   a) as heavy chain of the first full length antibody the amino        acid sequence of SEQ ID NO: 30, and as light chain of the first        full length antibody the amino acid sequence of SEQ ID NO: 31,        and    -   b) as heavy chain of the second full length antibody connected        to the light chain of the second full length antibody via a        peptide linker the amino acid sequence of SEQ ID NO: 29.

In one embodiment such bispecific, bivalent antibody is characterized incomprising

-   -   a) as heavy chain of the first full length antibody the amino        acid sequence of SEQ ID NO: 33, and as light chain of the first        full length antibody the amino acid sequence of SEQ ID NO: 34,        and    -   b) as heavy chain of the second full length antibody connected        to the light chain of the second full length antibody via a        peptide linker the amino acid sequence of SEQ ID NO: 32.

In one embodiment the antibody heavy chain variable domain (VH) and theantibody light chain variable domain (VL) of the heavy and light chainof the second full length antibody are disulfide stabilized byintroduction of a disulfide bond between the following positions: heavychain variable domain position 44 to light chain variable domainposition 100 (numbering always according to EU index of Kabat; (Kabat,E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed.,Public Health Service, National Institutes of Health, Bethesda, Md.(1991))). Such further disulfide stabilization is achieved by theintroduction of a disulfide bond between the variable domains VH and VLof the second full length antibody heavy and light chain. Techniques tointroduce unnatural disulfide bridges for stabilization are describede.g. in WO 94/029350, Rajagopal, V., et al, Prot. Engin. 10 (1997)1453-59; Kobayashi, et al., Nuclear Medicine & Biology, Vol. 25 (1998)387-393; or Schmidt, M., et al., Oncogene 18 (1999) 1711-1721.

Thus in one embodiment such bispecific, bivalent antibody ischaracterized in comprising a disulfide bond between the variabledomains of the second full length antibody heavy and light chain isbetween heavy chain variable domain position 44 and light chain variabledomain position 100, and comprises

-   -   a) as heavy chain of the first full length antibody the amino        acid sequence of SEQ ID NO: 36, and as light chain of the first        full length antibody the amino acid sequence of SEQ ID NO: 37,        and    -   b) as heavy chain of the second full length antibody connected        to the light chain of the second full length antibody via a        peptide linker the amino acid sequence of SEQ ID NO: 35.

In another aspect of the invention the bispecific antibody according tothe invention is characterized in comprising

-   -   a) the heavy chain and the light chain of a first full length        antibody that specifically binds to VEGF;

b) the heavy chain and the light chain of a second full length antibodythat specifically binds to ANG-2,

-   -   wherein the N-terminus of the heavy chain is connected to the        C-terminus of the light chain via a peptide linker; and wherein        the variable domains VL and VH are replaced by each other.

An exemplary scheme of this bispecific, bivalent antibody format forthis bispecific antibody specifically binding to human vascularendothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) isshown in FIG. 2 b including Knobs-into-Holes modified CH3 domains. Theantibodies based on this bispecific, bivalent antibody format are namedin the examples OAscXFab1.

In one embodiment such bispecific antibody is characterized incomprising

-   -   a) as heavy chain of the first full length antibody the SEQ ID        NO: 39, and as light chain of the first full length antibody the        SEQ ID NO: 40, and    -   b) as heavy chain of the second full length antibody connected        to the light chain of the second full length antibody via a        peptide linker the SEQ ID NO: 38.

In another aspect of the invention the bispecific antibody according tothe invention is characterized in comprising

-   -   a) the heavy chain and the light chain of a first full length        antibody that specifically binds to VEGF;    -   b) the heavy chain and the light chain of a second full length        antibody that specifically binds to ANG-2,    -   wherein the N-terminus of the heavy chain is connected to the        C-terminus of the light chain via a peptide linker; and wherein        the constant domains CL and CH1 are replaced by each other.

An exemplary scheme of this bispecific, bivalent antibody format forthis bispecific antibody specifically binding to human vascularendothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) isshown in FIG. 2 c including Knobs-into-Holes modified CH3 domains. Theantibodies based on this bispecific, bivalent antibody format are namedin the examples OAscXFab2 and OAscXFab3.

In one embodiment such bispecific antibody is characterized incomprising

-   -   a) as heavy chain of the first full length antibody the SEQ ID        NO: 42, and as light chain of the first full length antibody the        SEQ ID NO: 43, and    -   b) as heavy chain of the second full length antibody connected        to the light chain of the second full length antibody via a        peptide linker the SEQ ID NO: 41.

In one embodiment such bispecific antibody is characterized incomprising

-   -   a) as heavy chain of the first full length antibody the SEQ ID        NO: 45, and as light chain of the first full length antibody the        SEQ ID NO: 46, and    -   b) as heavy chain of the second full length antibody connected        to the light chain of the second full length antibody via a        peptide linker the SEQ ID NO: 44.

Accordingly one embodiment of the invention is a bispecific, bivalentantibody comprising a first antigen-binding site that specifically bindsto human VEGF and a second antigen-binding site that specifically bindsto human ANG-2, characterized in comprising the amino acid sequences ofSEQ ID NO: 29, of SEQ ID NO: 30, and of SEQ ID NO: 31.

Accordingly one embodiment of the invention is a bispecific, bivalentantibody comprising a first antigen-binding site that specifically bindsto human VEGF and a second antigen-binding site that specifically bindsto human ANG-2, characterized in comprising the amino acid sequences ofSEQ ID NO: 32, of SEQ ID NO: 33, and of SEQ ID NO: 34.

Accordingly one embodiment of the invention is a bispecific, bivalentantibody comprising a first antigen-binding site that specifically bindsto human VEGF and a second antigen-binding site that specifically bindsto human ANG-2, characterized in comprising the amino acid sequences ofSEQ ID NO: 35, of SEQ ID NO: 36, and of SEQ ID NO: 37.

Accordingly one embodiment of the invention is a bispecific, bivalentantibody comprising a first antigen-binding site that specifically bindsto human VEGF and a second antigen-binding site that specifically bindsto human ANG-2, characterized in comprising the amino acid sequences ofSEQ ID NO: 38, of SEQ ID NO: 39, and of SEQ ID NO: 40.

Accordingly one embodiment of the invention is a bispecific, bivalentantibody comprising a first antigen-binding site that specifically bindsto human VEGF and a second antigen-binding site that specifically bindsto human ANG-2, characterized in comprising the amino acid sequences ofSEQ ID NO: 41, of SEQ ID NO: 42, and of SEQ ID NO: 43.

Accordingly one embodiment of the invention is a bispecific, bivalentantibody comprising a first antigen-binding site that specifically bindsto human VEGF and a second antigen-binding site that specifically bindsto human ANG-2, characterized in comprising the amino acid sequences ofSEQ ID NO: 44, of SEQ ID NO: 45, and of SEQ ID NO: 46.

Preferably the CH3 domains of the bispecific, bivalent antibodyaccording to the invention is altered by the “knob-into-holes”technology which is described in detail with several examples in e.g. WO96/027011, Ridgway J. B., et al., Protein Eng 9 (1996) 617-621; andMerchant, A. M., et al., Nat Biotechnol 16 (1998) 677-681. In thismethod the interaction surfaces of the two CH3 domains are altered toincrease the heterodimerisation of both heavy chains containing thesetwo CH3 domains. Each of the two CH3 domains (of the two heavy chains)can be the “knob”, while the other is the “hole”. The introduction of adisulfide bridge stabilizes the heterodimers (Merchant, A. M, et al.,Nature Biotech 16 (1998) 677-681; Atwell, S., et al. J. Mol. Biol. 270(1997) 26-35) and increases the yield.

In a preferred aspect of the invention all bispecific antibodiesaccording to the invention are characterized in that

the CH3 domain of one heavy chain and the CH3 domain of the other heavychain each meet at an interface which comprises an original interfacebetween the antibody CH3 domains;wherein said interface is altered to promote the formation of thebispecific antibody, wherein the alteration is characterized in that:a) the CH3 domain of one heavy chain is altered,so that within the original interface the CH3 domain of one heavy chainthat meets the original interface of the CH3 domain of the other heavychain within the bispecific antibody,an amino acid residue is replaced with an amino acid residue having alarger side chain volume, thereby generating a protuberance within theinterface of the CH3 domain of one heavy chain which is positionable ina cavity within the interface of the CH3 domain of the other heavy chainandb) the CH3 domain of the other heavy chain is altered,so that within the original interface of the second CH3 domain thatmeets the original interface of the first CH3 domain within thebispecific antibody an amino acid residue is replaced with an amino acidresidue having a smaller side chain volume, thereby generating a cavitywithin the interface of the second CH3 domain within which aprotuberance within the interface of the first CH3 domain ispositionable.

Thus the antibody according to invention is preferably characterized inthat

-   -   the CH3 domain of the heavy chain of the full length antibody        of a) and the CH3 domain of the heavy chain of the full length        antibody of b) each meet at an interface which comprises an        alteration in the original interface between the antibody CH3        domains;    -   wherein i) in the CH3 domain of one heavy chain    -   an amino acid residue is replaced with an amino acid residue        having a larger side chain volume, thereby generating a        protuberance within the interface of the CH3 domain of one heavy        chain which is positionable in a cavity within the interface of        the CH3 domain of the other heavy chain    -   and wherein    -   ii) in the CH3 domain of the other heavy chain    -   an amino acid residue is replaced with an amino acid residue        having a smaller side chain volume, thereby generating a cavity        within the interface of the second CH3 domain within which a        protuberance within the interface of the first CH3 domain is        positionable.

Preferably said amino acid residue having a larger side chain volume isselected from the group consisting of arginine (R), phenylalanine (F),tyrosine (Y), tryptophan (W).

Preferably said amino acid residue having a smaller side chain volume isselected from the group consisting of alanine (A), serine (S), threonine(T), valine (V).

In one aspect of the invention both CH3 domains are further altered bythe introduction of cysteine (C) as amino acid in the correspondingpositions of each CH3 domain such that a disulfide bridge between bothCH3 domains can be formed.

In one embodiment, the bispecific antibody comprises a T366W mutation inthe CH3 domain of the “knobs chain” and T366S, L368A, Y407V mutations inthe CH3 domain of the “hole chain”. An additional interchain disulfidebridge between the CH3 domains can also be used (Merchant, A. M, et al.,Nature Biotech 16 (1998) 677-681) e.g. by introducing a Y349C mutationinto the CH3 domain of the “knobs chain” and a E356C mutation or a S354Cmutation into the CH3 domain of the “hole chain”.

In another embodiment, the bispecific antibody according to theinvention comprises Y349C, T366W mutations in one of the two CH3 domainsand E356C, T366S, L368A, Y407V mutations in the other of the two CH3domains. In a another preferred embodiment the bispecific antibodycomprises Y349C, T366W mutations in one of the two CH3 domains andS354C, T366S, L368A, Y407V mutations in the other of the two CH3 domains(the additional Y349C mutation in one CH3 domain and the additionalE356C or S354C mutation in the other CH3 domain forming a interchaindisulfide bridge) (numbering always according to EU index of Kabat;(Kabat, E. A., et al., Sequences of Proteins of Immunological Interest,5th ed., Public Health Service, National Institutes of Health, Bethesda,Md. (1991))). But also other knobs-in-holes technologies as described byEP 1 870 459 A1, can be used alternatively or additionally. Thus anotherexample for the bispecific antibody are R409D; K370E mutations in theCH3 domain of the “knobs chain” and D399K; E357K mutations in the CH3domain of the “hole chain” (numbering always according to EU index ofKabat; (Kabat, E. A., et al., Sequences of Proteins of ImmunologicalInterest, 5th ed., Public Health Service, National Institutes of Health,Bethesda, Md. (1991))).

In another embodiment the bispecific antibody comprises a T366W mutationin the CH3 domain of the “knobs chain” and T366S, L368A, Y407V mutationsin the CH3 domain of the “hole chain” and additionally R409D; K370Emutations in the CH3 domain of the “knobs chain” and D399K; E357Kmutations in the CH3 domain of the “hole chain”.

In another embodiment the bispecific antibody comprises Y349C, T366Wmutations in one of the two CH3 domains and S354C, T366S, L368A, Y407Vmutations in the other of the two CH3 domains or said trivalent,bispecific antibody comprises Y349C, T366W mutations in one of the twoCH3 domains and S354C, T366S, L368A, Y407V mutations in the other of thetwo CH3 domains and additionally R409D; K370E mutations in the CH3domain of the “knobs chain” and D399K; E357K mutations in the CH3 domainof the “hole chain”.

In one embodiment of the invention the bispecific antibody according tothe invention is characterized in having one or more of the followingproperties (determined in assays as described in Examples 3 to 7):

-   -   the bispecific, bivalent antibody binds to VEGF with a KD value        of the binding affinity of 5 nM or less;    -   the bispecific, bivalent antibody binds to ANG-2 with a KD value        of the binding affinity of 5 nM or less;    -   the bispecific, bivalent antibody inhibits ANG-2-induced Tie2        phosphorylation in HEK293 cells transfected with Tie2 with an        IC50 of 15 nM or less, (in one embodiment with an IC50 of 10 nM        or less);    -   the bispecific, bivalent antibody inhibits ANG-2 binding to Tie2        with an IC50 of 20 nM or less, (in one embodiment with an IC50        of 15 nM or less);    -   the bispecific, bivalent antibody inhibits VEGF binding to VEGF        receptor with an IC50 of 20 nM or less, (in one embodiment with        an IC50 of 15 nM or less);    -   the bispecific, bivalent antibody inhibits VEGF-induced        proliferation of

HUVEC cells with an with an IC50 of 10 nM or less, (in one embodimentwith an IC50 of 5 nM or less).

In one embodiment the bispecific, bivalent antibody is characterized incomprising

a first antigen-binding site that specifically binds to human VEGF and asecond antigen-binding site that specifically binds to human ANG-2,characterized in that

-   -   i) said first antigen-binding site comprises as heavy chain        variable domain (VH) the SEQ ID NO: 1, and as light chain        variable domain (VL) the SEQ ID NO: 2; and    -   ii) said second antigen-binding site comprises as heavy chain        variable domain (VH) the SEQ ID NO: 3, and as light chain        variable domain (VL) the SEQ ID NO: 4;        and having one or more of the following properties (determined        in assays as described in Examples 3 to 7):    -   the bispecific, bivalent antibody binds to VEGF with a KD value        of the binding affinity of 5 nM or less;    -   the bispecific, bivalent antibody binds to ANG-2 with a KD value        of the binding affinity of 5 nM or less;    -   the bispecific, bivalent antibody inhibits ANG-2-induced Tie2        phosphorylation in HEK293 cells transfected with Tie2 with an        1050 of 15 nM or less, (in one embodiment with an 1050 of 10 nM        or less);    -   the bispecific, bivalent antibody inhibits ANG-2 binding to Tie2        with an IC50 of 20 nM or less, (in one embodiment with an 1050        of 15 nM or less);    -   the bispecific, bivalent antibody inhibits VEGF binding to VEGF        receptor with an IC50 of 20 nM or less, (in one embodiment with        an IC50 of 15 nM or less);    -   the bispecific, bivalent antibody inhibits VEGF-induced        proliferation of HUVEC cells with an with an IC50 of 10 nM or        less, (in one embodiment with an IC50 of 5 nM or less).

In one aspect of the invention such bispecific antibody according to theinvention is characterized in comprising

-   -   a) the heavy chain and the light chain of a first full length        antibody that specifically binds to VEGF;

b) the modified heavy chain and modified light chain of a full lengthantibody that specifically binds to ANG-2, wherein the constant domainsCL and CH1 are replaced by each other;

and having one or more of the following properties (determined in assaysas described in Examples 3 to 7):

-   -   the bispecific, bivalent antibody binds to VEGF with a KD value        of the binding affinity of 5 nM or less;    -   the bispecific, bivalent antibody binds to ANG-2 with a KD value        of the binding affinity of 5 nM or less;    -   the bispecific, bivalent antibody inhibits ANG-2-induced Tie2        phosphorylation in HEK293 cells transfected with Tie2 with an        IC50 of 15 nM or less, (in one embodiment with an IC50 of 10 nM        or less);    -   the bispecific, bivalent antibody inhibits ANG-2 binding to Tie2        with an IC50 of 20 nM or less, (in one embodiment with an IC50        of 15 nM or less);    -   the bispecific, bivalent antibody inhibits VEGF binding to VEGF        receptor with an IC50 of 20 nM or less, (in one embodiment with        an IC50 of 15 nM or less);    -   the bispecific, bivalent antibody inhibits VEGF-induced        proliferation of

HUVEC cells with an with an IC50 of 10 nM or less, (in one embodimentwith an IC50 of 5 nM or less).

In one embodiment the bispecific, bivalent antibody is characterized incomprising

a first antigen-binding site that specifically binds to human VEGF and asecond antigen-binding site that specifically binds to human ANG-2,characterized in that

-   -   i) said first antigen-binding site comprises as heavy chain        variable domain (VH) the SEQ ID NO: 1 with no more than 1 amino        acid residue substitutions in the CDRs, and as light chain        variable domain (VL) the SEQ ID NO: 2 with no more than 1 amino        acid residue substitutions in the CDRs; and    -   ii) said second antigen-binding site comprises as heavy chain        variable domain (VH) the SEQ ID NO: 3 with no more than 1 amino        acid residue substitutions in the CDRs, and as light chain        variable domain (VL) the SEQ ID NO: 4 with no more than 1 amino        acid residue substitutions in the CDRs.

In one embodiment the bispecific, bivalent antibody is characterized incomprising

a first antigen-binding site that specifically binds to human VEGF and asecond antigen-binding site that specifically binds to human ANG-2,characterized in that

-   -   i) said first antigen-binding site comprises as heavy chain        variable domain (VH) the SEQ ID NO: 1 with no more than 1 amino        acid residue substitutions in the CDRs, and as light chain        variable domain (VL) the SEQ ID NO: 2 with no more than 1 amino        acid residue substitutions in the CDRs; and    -   ii) said second antigen-binding site comprises as heavy chain        variable domain (VH) the SEQ ID NO: 3 with no more than 1 amino        acid residue substitutions in the CDRs, and as light chain        variable domain (VL) the SEQ ID NO: 4 with no more than 1 amino        acid residue substitutions in the CDRs;        and having one or more of the following properties (determined        in assays as described in Examples 3 to 7):    -   the bispecific, bivalent antibody binds to VEGF with a KD value        of the binding affinity of 5 nM or less;    -   the bispecific, bivalent antibody binds to ANG-2 with a KD value        of the binding affinity of 5 nM or less;    -   the bispecific, bivalent antibody inhibits ANG-2-induced Tie2        phosphorylation in HEK293 cells transfected with Tie2 with an        IC50 of 15 nM or less, (in one embodiment with an IC50 of 10 nM        or less);    -   the bispecific, bivalent antibody inhibits ANG-2 binding to Tie2        with an IC50 of 20 nM or less, (in one embodiment with an IC50        of 15 nM or less);    -   the bispecific, bivalent antibody inhibits VEGF binding to VEGF        receptor with an IC50 of 20 nM or less, (in one embodiment with        an IC50 of 15 nM or less);    -   the bispecific, bivalent antibody inhibits VEGF-induced        proliferation of HUVEC cells with an with an IC50 of 10 nM or        less, (in one embodiment with an IC50 of 5 nM or less).

In one aspect of the invention the bispecific antibody according to theinvention is characterized in comprising

-   -   a) the heavy chain and the light chain of a first full length        antibody that specifically binds to VEGF,    -   and wherein the heavy chain of the first full length antibody        comprises the amino acid sequence of SEQ ID NO:7 with no more        than 1 amino acid residue substitutions in the CDRs, and the        light chain of the first full length antibody comprises the        amino acid sequence of SEQ ID NO: 5 with no more than 1 amino        acid residue substitutions in the CDRs, and    -   b) the modified heavy chain and modified light chain of a full        length antibody that specifically binds to ANG-2, wherein the        constant domains CL and CH1 are replaced by each other,    -   and wherein the modified heavy chain of the second full length        antibody comprises the amino acid sequence of SEQ ID NO: 8 with        no more than 1 amino acid residue substitutions in the CDRs, and        the modified light chain of the second full length antibody        comprises the amino acid sequence of SEQ ID NO: 6 with no more        than 1 amino acid residue substitutions in the CDRs.

In one aspect of the invention the bispecific antibody according to theinvention is characterized in comprising

-   -   a) the heavy chain and the light chain of a first full length        antibody that specifically binds to VEGF,    -   and wherein the heavy chain of the first full length antibody        comprises the amino acid sequence of SEQ ID NO:7 with no more        than 1 amino acid residue substitutions in the CDRs, and the        light chain of the first full length antibody comprises the        amino acid sequence of SEQ ID NO: 5 with no more than 1 amino        acid residue substitutions in the CDRs, and    -   b) the modified heavy chain and modified light chain of a full        length antibody that specifically binds to ANG-2, wherein the        constant domains CL and CH1 are replaced by each other,    -   and wherein the modified heavy chain of the second full length        antibody comprises the amino acid sequence of SEQ ID NO: 8 with        no more than 1 amino acid residue substitutions in the CDRs, and        the modified light chain of the second full length antibody        comprises the amino acid sequence of SEQ ID NO: 6 with no more        than 1 amino acid residue substitutions in the CDRs;        and having one or more of the following properties (determined        in assays as described in Examples 3 to 7):    -   the bispecific, bivalent antibody binds to VEGF with a KD value        of the binding affinity of 5 nM or less;    -   the bispecific, bivalent antibody binds to ANG-2 with a KD value        of the binding affinity of 5 nM or less;    -   the bispecific, bivalent antibody inhibits ANG-2-induced Tie2        phosphorylation in HEK293 cells transfected with Tie2 with an        1050 of 15 nM or less, (in one embodiment with an 1050 of 10 nM        or less);    -   the bispecific, bivalent antibody inhibits ANG-2 binding to Tie2        with an 1050 of 20 nM or less, (in one embodiment with an 1050        of 15 nM or less);    -   the bispecific, bivalent antibody inhibits VEGF binding to VEGF        receptor with an 1050 of 20 nM or less, (in one embodiment with        an 1050 of 15 nM or less);    -   the bispecific, bivalent antibody inhibits VEGF-induced        proliferation of HUVEC cells with an with an IC50 of 10 nM or        less, (in one embodiment with an 1050 of 5 nM or less).

As used herein, “antibody” refers to a binding protein that comprisesantigen-binding sites. The terms “binding site” or “antigen-bindingsite” as used herein denotes the region(s) of an antibody molecule towhich a ligand actually binds. The term “antigen-binding site” comprisesan antibody heavy chain variable domains (VH) and an antibody lightchain variable domains (VL) (pair of VH/VL).).

Antibody specificity refers to selective recognition of the antibody fora particular epitope of an antigen. Natural antibodies, for example, aremonospecific.

“Bispecific antibodies” according to the invention are antibodies whichhave two different antigen-binding specificities. Antibodies of thepresent invention are specific for two different antigens, VEGF as firstantigen and ANG-2 as second antigen.

The term “monospecific” antibody as used herein denotes an antibody thathas one or more binding sites each of which bind to the same epitope ofthe same antigen.

The term “valent” as used within the current application denotes thepresence of a specified number of binding sites in an antibody molecule.As such, the terms “bivalent”, “tetravalent”, and “hexavalent” denotethe presence of two binding site, four binding sites, and six bindingsites, respectively, in an antibody molecule. The bispecific antibodiesaccording to the invention are “bivalent”.

The term “VEGF” as used herein refers to human vascular endothelialgrowth factor (VEGF/VEGF-A) (SEQ ID NO: 47) which is described e.g. inLeung, D. W., et al., Science 246 (1989) 1306-9; Keck, P. J., et al.,Science 246 (1989) 1309-12 and Connolly, D. T., et al., J. Biol. Chem.264 (1989) 20017-24. VEGF is involved in the regulation of normal andabnormal angiogenesis and neovascularization associated with tumors andintraocular disorders (Ferrara, N., et al., Endocr. Rev. 18 (1997) 4-25;Berkman, R. A., et al., J. Clin. Invest. 91 (1993) 153-159; Brown, L.F., et al., Human Pathol. 26 (1995) 86-91; Brown, L. F., et al., CancerRes. 53 (1993) 4727-4735; Mattern, J., et al., Brit. J. Cancer. 73(1996) 931-934; and Dvorak, H. F., et al., Am. J. Pathol. 146 (1995)1029-1039). VEGF is a homodimeric glycoprotein that has been isolatedfrom several sources. VEGF shows highly specific mitogenic activity forendothelial cells.

The term “ANG-2” as used herein refers to human angiopoietin-2 (ANG-2)(alternatively abbreviated with ANGPT2 or ANG2) (SEQ ID NO: 48) which isdescribed e.g. in Maisonpierre, P. C., et al, Science 277 (1997) 55-60and Cheung, A. H., et al., Genomics 48 (1998) 389-91. Theangiopoietins-1 and -2 were discovered as ligands for the Ties, a familyof tyrosine kinases that is selectively expressed within the vascularendothelium. Yancopoulos, G. D., et al., Nature 407 (2000) 242-48. Thereare now four definitive members of the angiopoietin family.Angiopoietin-3 and -4 (Ang-3 and Ang-4) may represent widely divergedcounterparts of the same gene locus in mouse and man. Kim, I., et al.,FEBS Let, 443 (1999) 353-56; Kim, 1., et al., J Biol Chem 274 (1999)26523-28. ANG-1 and ANG-2 were originally identified in tissue cultureexperiments as agonist and antagonist, respectively (see for ANG-1:Davis, S., et al., Cell 87 (1996) 1161-69; and for ANG-2: Maisonpierre,P. C., et al., Science 277 (1997) 55-60) All of the known angiopoietinsbind primarily to Tie2, and both Ang-1 and -2 bind to Tie2 with anaffinity of 3 nM (Kd). Maisonpierre, P. C., et al., Science 277 (1997)55-60.

An antigen-binding sites of the bispecific antibody of the inventioncontain six complementarity determining regions (CDRs) which contributein varying degrees to the affinity of the binding site for antigen.There are three heavy chain variable domain CDRs (CDRH1, CDRH2 andCDRH3) and three light chain variable domain CDRs (CDRL1, CDRL2 andCDRL3). The extent of CDR and framework regions (FRs) is determined bycomparison to a compiled database of amino acid sequences in which thoseregions have been defined according to variability among the sequences.Also included within the scope of the invention are functional antigenbinding sites comprised of fewer CDRs (i.e., where binding specificityis determined by three, four or five CDRs). For example, less than acomplete set of 6 CDRs may be sufficient for binding. In some cases, aVH or a VL domain will be sufficient.

The antibodies of the invention further comprise immunoglobulin constantregions of one or more immunoglobulin classes. Immunoglobulin classesinclude IgG, IgM, IgA, IgD, and IgE isotypes and, in the case of IgG andIgA, their subtypes.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of a singleamino acid composition.

The term “chimeric antibody” refers to an antibody comprising a variableregion, i.e., binding region, from one source or species and at least aportion of a constant region derived from a different source or species,usually prepared by recombinant DNA techniques. Chimeric antibodiescomprising a murine variable region and a human constant region arepreferred. Other preferred forms of “chimeric antibodies” encompassed bythe present invention are those in which the constant region has beenmodified or changed from that of the original antibody to generate theproperties according to the invention, especially in regard to C1qbinding and/or Fc receptor (FcR) binding. Such chimeric antibodies arealso referred to as “class-switched antibodies.”. Chimeric antibodiesare the product of expressed immunoglobulin genes comprising DNAsegments encoding immunoglobulin variable regions and DNA segmentsencoding immunoglobulin constant regions. Methods for producing chimericantibodies involve conventional recombinant DNA and gene transfectiontechniques are well known in the art. See, e.g., Morrison, S. L., etal., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; U.S. Pat. No.5,202,238 and U.S. Pat. No. 5,204,244.

The term “humanized antibody” refers to antibodies in which theframework or “complementarity determining regions” (CDR) have beenmodified to comprise the CDR of an immunoglobulin of differentspecificity as compared to that of the parent immunoglobulin. In apreferred embodiment, a murine CDR is grafted into the framework regionof a human antibody to prepare the “humanized antibody.” See, e.g.,Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M. S.,et al., Nature 314 (1985) 268-270. Particularly preferred CDRscorrespond to those representing sequences recognizing the antigensnoted above for chimeric antibodies. Other forms of “humanizedantibodies” encompassed by the present invention are those in which theconstant region has been additionally modified or changed from that ofthe original antibody to generate the properties according to theinvention, especially in regard to Clq binding and/or Fc receptor (FcR)binding.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well-known in thestate of the art (van Dijk, M. A., and van de Winkel, J. G., Curr. Opin.Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced intransgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire or a selection of human antibodies in theabsence of endogenous immunoglobulin production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge(see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993)2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258;Brueggemann, M., et al., Year Immunol. 7 (1993) 33-40). Human antibodiescan also be produced in phage display libraries (Hoogenboom, H. R., andWinter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J. D., et al., J.Mol. Biol. 222 (1991) 581-597). The techniques of Cole, A., et al. andBoerner, P., et al. are also available for the preparation of humanmonoclonal antibodies (Cole, A., et al., Monoclonal Antibodies andCancer Therapy, Liss, A. L., p. 77 (1985); and Boerner, P., et al., J.Immunol. 147 (1991) 86-95). As already mentioned for chimeric andhumanized antibodies according to the invention the term “humanantibody” as used herein also comprises such antibodies which aremodified in the constant region to generate the properties according tothe invention, especially in regard to Clq binding and/or FcR binding,e.g. by “class switching” i.e. change or mutation of Fc parts (e.g. fromIgG1 to IgG4 and/or IgG1/IgG4 mutation).

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies isolated from a hostcell such as a NS0 or CHO cell or from an animal (e.g. a mouse) that istransgenic for human immunoglobulin genes or antibodies expressed usinga recombinant expression vector transfected into a host cell. Suchrecombinant human antibodies have variable and constant regions in arearranged form. The recombinant human antibodies according to theinvention have been subjected to in vivo somatic hypermutation. Thus,the amino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangerm line VH and VL sequences, may not naturally exist within the humanantibody germ line repertoire in vivo.

The “variable domain” (variable domain of a light chain (VL), variabledomain of a heavy chain (VH) as used herein denotes each of the pair oflight and heavy chains which is involved directly in binding theantibody to the antigen. The domains of variable human light and heavychains have the same general structure and each domain comprises fourframework (FR) regions whose sequences are widely conserved, connectedby three “hypervariable regions” (or complementarity determiningregions, CDRs). The framework regions adopt a β-sheet conformation andthe CDRs may form loops connecting the β-sheet structure. The CDRs ineach chain are held in their three-dimensional structure by theframework regions and form together with the CDRs from the other chainthe antigen binding site. The antibody heavy and light chain CDR3regions play a particularly important role in the bindingspecificity/affinity of the antibodies according to the invention andtherefore provide a further object of the invention.

The terms “hypervariable region” or “antigen-binding portion of anantibody” when used herein refer to the amino acid residues of anantibody which are responsible for antigen-binding. The hypervariableregion comprises amino acid residues from the “complementaritydetermining regions” or “CDRs”. “Framework” or “FR” regions are thosevariable domain regions other than the hypervariable region residues asherein defined. Therefore, the light and heavy chains of an antibodycomprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. CDRs on each chain are separated by such framework aminoacids. Especially, CDR3 of the heavy chain is the region whichcontributes most to antigen binding. CDR and FR regions are determinedaccording to the standard definition of Kabat, E. A., et al., Sequencesof Proteins of Immunological Interest, 5th ed., Public Health Service,National Institutes of Health, Bethesda, Md. (1991) (includes thenumbering according to the EU Index of Kabat, (abbreviated as numberingaccording to Kabat herein below)).

As used herein, the term “binding” or “specifically binding” refers tothe binding of the antibody to an epitope of the antigen (either humanVEGF or human ANG-2) in an in vitro assay, preferably in an plasmonresonance assay (BIAcore, GE-Healthcare Uppsala, Sweden) (Example 3)with purified wild-type antigen. The affinity of the binding is definedby the terms ka (rate constant for the association of the antibody fromthe antibody/antigen complex), k_(D) (dissociation constant), and K_(D)(k_(D)/ka). In one embodiment binding or specifically binding means abinding affinity (K_(D)) of 10⁻⁸ mol/l or less, preferably 10⁻⁹ M to10⁻¹³ mol/l.

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an antibody. In certain embodiments, epitopedeterminant include chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, incertain embodiments, may have specific three dimensional structuralcharacteristics, and or specific charge characteristics. An epitope is aregion of an antigen that is bound by an antibody.

In certain embodiments, an antibody is said to specifically bind anantigen when it preferentially recognizes its target antigen in acomplex mixture of proteins and/or macromolecules.

The term “full length antibody” denotes an antibody consisting of two“full length antibody heavy chains” and two “full length antibody lightchains” (see FIG. 1). A “full length antibody heavy chain” is apolypeptide consisting in N-terminal to C-terminal direction of anantibody heavy chain variable domain (VH), an antibody constant heavychain domain 1 (CH1), an antibody hinge region (HR), an antibody heavychain constant domain 2 (CH2), and an antibody heavy chain constantdomain 3 (CH3), abbreviated as VH-CH1-HR-CH2-CH3; and optionally anantibody heavy chain constant domain 4 (CH4) in case of an antibody ofthe subclass IgE. Preferably the “full length antibody heavy chain” is apolypeptide consisting in N-terminal to C-terminal direction of VH, CH1,HR, CH2 and CH3. A “full length antibody light chain” is a polypeptideconsisting in N-terminal to C-terminal direction of an antibody lightchain variable domain (VL), and an antibody light chain constant domain(CL), abbreviated as VL-CL. The antibody light chain constant domain(CL) can be κ (kappa) or λ (lambda). The two full length antibody chainsare linked together via inter-polypeptide disulfide bonds between the CLdomain and the CH1 domain and between the hinge regions of the fulllength antibody heavy chains. Examples of typical full length antibodiesare natural antibodies like IgG (e.g. IgG 1 and IgG2), IgM, IgA, IgD,and IgE. The full length antibodies according to the invention can befrom a single species e.g. human, or they can be chimerized or humanizedantibodies. The full length antibodies according to the inventioncomprise two antigen binding sites each formed by a pair of VH and VL,which both specifically bind to the same antigen. The C-terminus of theheavy or light chain of said full length antibody denotes the last aminoacid at the C-terminus of said heavy or light chain. The N-terminus ofthe heavy or light chain of said full length antibody denotes the lastamino acid at the N-terminus of said heavy or light chain.

The term “peptide linker” as used within the invention denotes a peptidewith amino acid sequences, which is preferably of synthetic origin.These peptides according to invention are used to connect the C-terminusof the light chain to the N-terminus of heavy chain of the second fulllength antibody (that specifically binds to a second antigen) via apeptide linker. The peptide linker within the second full lengthantibody heavy and light chain is a peptide with an amino acid sequencewith a length of at least 30 amino acids, preferably with a length of 32to 50 amino acids. In one the peptide linker is a peptide with an aminoacid sequence with a length of 32 to 40 amino acids. In one embodimentsaid linker is (GxS)n with G=glycine, S=serine, (x=3, n=8, 9 or 10 andm=0, 1, 2 or 3) or (x=4 and n=6, 7 or 8 and m=0, 1, 2 or 3), preferablywith x=4, n=6 or 7 and m=0, 1, 2 or 3, more preferably with x=4, n=7 andm=2. In one embodiment said linker is (G₄S)₆G₂.

The term “constant region” as used within the current applicationsdenotes the sum of the domains of an antibody other than the variableregion. The constant region is not involved directly in binding of anantigen, but exhibits various effector functions. Depending on the aminoacid sequence of the constant region of their heavy chains, antibodiesare divided in the classes: IgA, IgD, IgE, IgG and IgM, and several ofthese may be further divided into subclasses, such as IgG1, IgG2, IgG3,and IgG4, IgA1 and IgA2. The heavy chain constant regions thatcorrespond to the different classes of antibodies are called α, δ, ε, γ,and μ, respectively. The light chain constant regions which can be foundin all five antibody classes are called κ (kappa) and λ (lambda).

The term “constant region derived from human origin” as used in thecurrent application denotes a constant heavy chain region of a humanantibody of the subclass IgG1, IgG2, IgG3, or IgG4 and/or a constantlight chain kappa or lambda region. Such constant regions are well knownin the state of the art and e.g. described by Kabat, E. A., (see e.g.Johnson, G., and Wu, T. T., Nucleic Acids Res. 28 (2000) 214-218; Kabat,E. A., et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788).

Preferably the bispecific, bivalent antibodies according to theinvention have a constant region of human IgG1 subclass.

While antibodies of the IgG4 subclass show reduced Fc receptor(FcγRIIIa) binding, antibodies of other IgG subclasses show binding.However Pro238, Asp265, Asp270, Asn297 (loss of Fc carbohydrate),Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254, Lys288, Thr307,Gln311, Asn434, and His435 are residues which, if altered, provide alsoreduced Fc receptor binding (Shields, R. L., et al., J. Biol. Chem. 276(2001) 6591-6604; Lund, J., et al., FASEB J. 9 (1995) 115-119; Morgan,A., et al., Immunology 86 (1995) 319-324; EP 0 307 434).

In one embodiment an antibody according to the invention has a reducedFcR binding compared to an IgG1 antibody and the bispecific, bivalentantibody is in regard to FcR binding of IgG4 subclass or of IgG1subclass with a mutation in S228, L234, L235 and/or D265, and/orcontains the PVA236 mutation. In one embodiment the mutations in thebispecific bivalent antibody are in IgG4 S228P and L235E and in IgG1L234A and L235A.

Another aspect of the invention a bispecific, bivalent antibodycharacterized in comprising

-   -   a) the heavy chain and the light chain of a first full length        antibody that specifically binds to a first antigen;

b) the heavy chain and the light chain of a second full length antibodythat specifically binds to a second antigen,

-   -   wherein the N-terminus of the heavy chain is connected to the        C-terminus of the light chain via a peptide linker; and    -   wherein the variable domains VL and VH or the constant domains        CL and CH1 are replaced by each other.

Preferably the CH3 domains of this bispecific, bivalent antibody formatis altered by the “knob-into-holes” technology which is described indetail with several examples in e.g. WO 96/027011, Ridgway J. B., etal., Protein Eng 9 (1996) 617-621; and Merchant, A. M., et al., NatBiotechnol 16 (1998) 677-681. In this method the interaction surfaces ofthe two CH3 domains are altered to increase the heterodimerisation ofboth heavy chains containing these two CH3 domains. Each of the two CH3domains (of the two heavy chains) can be the “knob”, while the other isthe “hole”. The introduction of a disulfide bridge stabilizes theheterodimers (Merchant, A. M, et al., Nature Biotech 16 (1998) 677-681;Atwell, S., et al. J. Mol. Biol. 270 (1997) 26-35) and increases theyield. For further details and embodiments see above.

Another aspect of the invention a bispecific, bivalent antibodycharacterized in comprising

-   -   a) the heavy chain and the light chain of a first full length        antibody that specifically binds to a first antigen;

b) the heavy chain and the light chain of a second full length antibodythat specifically binds to a second antigen,

-   -   wherein the N-terminus of the heavy chain is connected to the        C-terminus of the light chain via a peptide linker; and    -   wherein the variable domains VL and VH are replaced by each        other.

An exemplary scheme of this bispecific, bivalent antibody format isshown in FIG. 2 b including Knobs-into-Holes modified CH3 domains. Theantibodies based on this bispecific, bivalent antibody format are namedin the examples OAscXFab1.

In one embodiment such bispecific antibody is characterized incomprising

-   -   a) as heavy chain of the first full length antibody the SEQ ID        NO: 39, and as light chain of the first full length antibody the        SEQ ID NO: 40, and    -   b) as heavy chain of the second full length antibody connected        to the light chain of the second full length antibody via a        peptide linker the SEQ ID NO: 38.

Another aspect of the invention a bispecific, bivalent antibodycharacterized in comprising

-   -   a) the heavy chain and the light chain of a first full length        antibody that specifically binds to a first antigen;    -   b) the heavy chain and the light chain of a second full length        antibody that specifically binds to a second antigen,    -   wherein the N-terminus of the heavy chain is connected to the        C-terminus of the light chain via a peptide linker; and    -   wherein the constant domains CL and CH1 are replaced by each        other.

An exemplary scheme of this bispecific, bivalent antibody format isshown in FIG. 2 c including Knobs-into-Holes modified CH3 domains. Theantibodies based on this bispecific, bivalent antibody format are namedin the examples OAscXFab2 and OAscXFab3.

In one embodiment such bispecific antibody is characterized incomprising

-   -   a) as heavy chain of the first full length antibody the SEQ ID        NO: 42, and as light chain of the first full length antibody the        SEQ ID NO: 43, and    -   b) as heavy chain of the second full length antibody connected        to the light chain of the second full length antibody via a        peptide linker the SEQ ID NO: 41.

In one embodiment such bispecific antibody is characterized incomprising

-   -   a) as heavy chain of the first full length antibody the SEQ ID        NO: 45, and as light chain of the first full length antibody the        SEQ ID NO: 46, and    -   b) as heavy chain of the second full length antibody connected        to the light chain of the second full length antibody via a        peptide linker the SEQ ID NO: 44.

The antibody according to the invention is produced by recombinantmeans. Thus, one aspect of the current invention is a nucleic acidencoding the antibody according to the invention and a further aspect isa cell comprising said nucleic acid encoding an antibody according tothe invention. Methods for recombinant production are widely known inthe state of the art and comprise protein expression in prokaryotic andeukaryotic cells with subsequent isolation of the antibody and usuallypurification to a pharmaceutically acceptable purity. For the expressionof the antibodies as aforementioned in a host cell, nucleic acidsencoding the respective modified light and heavy chains are insertedinto expression vectors by standard methods. Expression is performed inappropriate prokaryotic or eukaryotic host cells like CHO cells, NS0cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast, or E.coli cells, and the antibody is recovered from the cells (supernatant orcells after lysis). General methods for recombinant production ofantibodies are well-known in the state of the art and described, forexample, in the review articles of Makrides, S. C., Protein Expr. Purif.17 (1999) 183-202; Geisse, S., et al., Protein Expr. Purif. 8 (1996)271-282; Kaufman, R. J., Mol. Biotechnol. 16 (2000) 151-160; Werner, R.G., Drug Res. 48 (1998) 870-880.

Accordingly one embodiment of the invention is a method for thepreparation of a bispecific antibody according to the invention,comprising the steps of

-   -   a) transforming a host cell with vectors comprising nucleic acid        molecules encoding said antibody;    -   b) culturing the host cell under conditions that allow synthesis        of said antibody molecule; and    -   c) recovering said antibody molecule from said culture.

The bispecific antibodies are suitably separated from the culture mediumby conventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography. DNA and RNAencoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures. The hybridoma cells can serve as a sourceof such DNA and RNA. Once isolated, the DNA may be inserted intoexpression vectors, which are then transfected into host cells such asHEK 293 cells, CHO cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of recombinantmonoclonal antibodies in the host cells.

Amino acid sequence variants (or mutants) of the bispecific antibody areprepared by introducing appropriate nucleotide changes into the antibodyDNA, or by nucleotide synthesis. Such modifications can be performed,however, only in a very limited range. For example, the modifications donot alter the above mentioned antibody characteristics such as the IgGisotype and antigen binding, but may improve the yield of therecombinant production, protein stability or facilitate thepurification.

The term “host cell” as used in the current application denotes any kindof cellular system which can be engineered to generate the antibodiesaccording to the current invention. In one embodiment HEK293 cells andCHO cells are used as host cells. As used herein, the expressions“cell,” “cell line,” and “cell culture” are used interchangeably and allsuch designations include progeny. Thus, the words “transformants” and“transformed cells” include the primary subject cell and culturesderived therefrom without regard for the number of transfers. It is alsounderstood that all progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations. Variant progenythat have the same function or biological activity as screened for inthe originally transformed cell are included.

Expression in NS0 cells is described by, e.g., Barnes, L. M., et al.,Cytotechnology 32 (2000) 109-123; Barnes, L. M., et al., Biotech.Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g.,Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9. Cloning ofvariable domains is described by Orlandi, R., et al., Proc. Natl. Acad.Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci.USA 89 (1992) 4285-4289; and Norderhaug, L., et al., J. Immunol. Methods204 (1997) 77-87. A preferred transient expression system (HEK 293) isdescribed by Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30(1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996)191-199.

The control sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters, enhancersand polyadenylation signals.

A nucleic acid is “operably linked” when it is placed in a functionalrelationship with another nucleic acid sequence. For example, DNA for apre-sequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a pre-protein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading frame. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

Purification of antibodies is performed in order to eliminate cellularcomponents or other contaminants, e.g. other cellular nucleic acids orproteins, by standard techniques, including alkaline/SDS treatment, CsClbanding, column chromatography, agarose gel electrophoresis, and otherswell known in the art. See Ausubel, F., et al., ed. Current Protocols inMolecular Biology, Greene Publishing and Wiley Interscience, New York(1987). Different methods are well established and widespread used forprotein purification, such as affinity chromatography with microbialproteins (e.g. protein A or protein G affinity chromatography), ionexchange chromatography (e.g. cation exchange (carboxymethyl resins),anion exchange (amino ethyl resins) and mixed-mode exchange), thiophilicadsorption (e.g. with beta-mercaptoethanol and other SH ligands),hydrophobic interaction or aromatic adsorption chromatography (e.g. withphenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic acid),metal chelate affinity chromatography (e.g. with Ni(II)- andCu(II)-affinity material), size exclusion chromatography, andelectrophoretical methods (such as gel electrophoresis, capillaryelectrophoresis) (Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75(1998) 93-102).

It has now been found that the bispecific antibodies against human VEGFand human ANG-2 according to the current invention have valuablecharacteristics such as high stability and valuablepharmacokinetic/pharmacodynamic properties like e.g. good (i.e. slow)clearance (e.g. at low doses).

The bispecific, bivalent antibodies according to the invention showbenefits for human patients in need of a VEGF and ANG-2 targetingtherapy.

Furthermore they have biological or pharmacological activity and show invivo tumor growth inhibition and/or inhibition of tumor angiogenesis.

The bispecific antibodies according to the invention are highlyeffective in

-   -   a) tumor growth inhibition (e.g. with the bispecific antibodies        according to the invention tumor stasis could be achieved        already at lower concentrations compared to the combination of        the two monospecific antibodies (e.g in the COLO205 and the KPL4        tumor models of Example 9 and 10, tumor stasis was already        achieved with 10 mg/kg XMAb1 compared to the combination of 10        mg/kg of ANG2i-LC06+10 mg/kg of Avastin), and/or    -   b) inhibition of tumor angiogenesis or vascular diseases (e.g.        maximal antiangiogenic effects with the bispecific antibodies        according to the invention could already be achieved at lower        concentrations compared to the combination of the two        monospecific antibodies (e.g. in the mouse corneal angiogenesis        assay of Example 8, the maximal antiangiogenic effect was        already achieved with 10 mg/kg XMAb1 compared to the combination        of 10 mg/kg of ANG2i-LC06+10 mg/kg of Avastin).

Finally the bivalent bispecific against human VEGF and human ANG-2according to the current invention may have a valuable efficacy/toxicityprofile and may provide benefits for a patient in the need of ananti-VEGF and anti-ANG-2 therapy.

One aspect of the invention is a pharmaceutical composition comprisingan antibody according to the invention. Another aspect of the inventionis the use of an antibody according to the invention for the manufactureof a pharmaceutical composition. A further aspect of the invention is amethod for the manufacture of a pharmaceutical composition comprising anantibody according to the invention. In another aspect, the presentinvention provides a composition, e.g. a pharmaceutical composition,containing an antibody according to the present invention, formulatedtogether with a pharmaceutical carrier.

One embodiment of the invention is the bispecific antibody according tothe invention for the treatment of cancer.

Another aspect of the invention is said pharmaceutical composition forthe treatment of cancer.

Another aspect of the invention is the use of an antibody according tothe invention for the manufacture of a medicament for the treatment ofcancer.

Another aspect of the invention is method of treatment of patientsuffering from cancer by administering an antibody according to theinvention to a patient in the need of such treatment.

Another aspect of the invention is said pharmaceutical composition forthe prevention of metastasis.

The invention comprises the bispecific antibody according to theinvention for the prevention of metastasis.

Another aspect of the invention is the use of a bispecific antibodyaccording to the invention for the manufacture of a medicament for theprevention of metastasis.

Another aspect of the invention is a method of prevention metastasis inpatient suffering from primary cancer by administering a bispecificaccording to the invention to a patient in the need of such preventativetreatment.

We could show highly efficient prevention of spontaneousmetastasis/secondary tumors in vivo in a orthotopic and a subcutaneouscancer model (see Example 9) (in contrast to experimental model wherethe tumor cells are injected i.v. This is similar to the clinicalsituation wherein cells disseminate from a primary tumor and metastaseto secondary organ like lung or liver (where secondary tumors).

The term “metastasis” according to the invention refers to thetransmission of cancerous cells from the primary tumor to one or moresites elsewhere in a patient where then secondary tumors develop. Meansto determine if a cancer has metastasized are known in the art andinclude bone scan, chest X-ray, CAT scan, MRI scan, and tumor markertests.

The term “prevention of metastasis” or “prevention of secondary tumors”as used herein have the same meaning and refers a prophylactic agentagainst metastasis in patient suffering from cancer in this wayinhibiting or reducing a further transmission of cancerous cells fromthe primary tumor to one or more sites elsewhere in a patient. Thismeans that the metastasis of the primary, tumor or cancer is prevented,delayed, or reduced and thus the development of secondary tumors isprevented, delayed, or reduced. Preferably the metastasis i.e. secondarytumors of the lung are prevented or reduced, which means that metastatictransmission of cancerous cells from the primary tumor to the lung isprevented or reduced.

As used herein, “pharmaceutical carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g. by injection or infusion).

A composition of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. To administer a compound of the invention bycertain routes of administration, it may be necessary to coat thecompound with, or co-administer the compound with, a material to preventits inactivation. For example, the compound may be administered to asubject in an appropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Pharmaceutical carriers include sterile aqueous solutions ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intra-arterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtrachcal, subcutaneous, subcuticular, intra-articular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

The term cancer as used herein refers to proliferative diseases, such aslymphomas, lymphocytic leukemias, lung cancer, non small cell lung(NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,cancer of the anal region, stomach cancer, gastric cancer, colon cancer,breast cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, Hodgkin's Disease, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, prostate cancer, cancer of the bladder,cancer of the kidney or ureter, renal cell carcinoma, carcinoma of therenal pelvis, mesothelioma, hepatocellular cancer, biliary cancer,neoplasms of the central nervous system (CNS), spinal axis tumors, brainstem glioma, glioblastoma multiforme, astrocytomas, schwanomas,cpendymonas, medulloblastomas, meningiomas, squamous cell carcinomas,pituitary adenoma and Ewings sarcoma, including refractory versions ofany of the above cancers, or a combination of one or more of the abovecancers.

Another aspect of the invention is the bispecific antibody according tothe invention or said pharmaceutical composition as anti-angiogenicagent. Such anti-angiogenic agent can be used for the treatment ofcancer, especially solid tumors, and other vascular diseases.

One embodiment of the invention is the bispecific antibody according tothe invention for the treatment of vascular diseases.

Another aspect of the invention is said pharmaceutical composition forthe treatment of vascular diseases.

Another aspect of the invention is the use of an antibody according tothe invention for the manufacture of a medicament for the treatment ofvascular diseases.

Another aspect of the invention is method of treatment of patientsuffering from vascular diseases by administering an antibody accordingto the invention to a patient in the need of such treatment.

The term “vascular diseases” includes Cancer, Inflammatory diseases,Atherosclerosis, Ischemia, Trauma, Sepsis, COPD, Asthma, Diabetes, AMD,Retinopathy, Stroke, Adipositas, Acute lung injury, Hemorrhage, Vascularleak e.g. Cytokine induced, Allergy, Graves' Disease, Hashimoto'sAutoimmune Thyroiditis, Idiopathic Thrombocytopenic Purpura, Giant CellArteritis, Rheumatoid Arthritis, Systemic Lupus Erythematosus (SLE),Lupus Nephritis, Crohn's Disease, Multiple Sclerosis, UlcerativeColitis, especially to solid tumors, intraocular neovascular syndromessuch as proliferative retinopathies or age-related macular degeneration(AMD), rheumatoid arthritis, and psoriasis (Folkman, J., et al., J.Biol. Chem. 267 (1992) 10931-10934; Klagsbrun, M., et al., Annu. Rev.Physiol. 53 (1991) 217-239; and Garner, A., Vascular diseases, In:Pathobiology of ocular disease, A dynamic approach, Garner, A., andKlintworth, G. K., (eds.), 2nd edition, Marcel Dekker, New York (1994),pp 1625-1710).

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol, sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts.

The composition must be sterile and fluid to the extent that thecomposition is deliverable by syringe. In addition to water, the carrierpreferably is an isotonic buffered saline solution.

Proper fluidity can be maintained, for example, by use of coating suchas lecithin, by maintenance of required particle size in the case ofdispersion and by use of surfactants. In many cases, it is preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol or sorbitol, and sodium chloride in the composition.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Variant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

The term “transformation” as used herein refers to process of transferof a vectors/nucleic acid into a host cell. If cells without formidablecell wall barriers are used as host cells, transfection is carried oute.g. by the calcium phosphate precipitation method as described byGraham, F. L., van der Eb, A. J., Virology 52 (1973) 546-467. However,other methods for introducing DNA into cells such as by nuclearinjection or by protoplast fusion may also be used. If prokaryotic cellsor cells which contain substantial cell wall constructions are used,e.g. one method of transfection is calcium treatment using calciumchloride as described by Cohen, S. N., et al., PNAS. 69 (1972)2110-2114.

As used herein, “expression” refers to the process by which a nucleicacid is transcribed into mRNA and/or to the process by which thetranscribed mRNA (also referred to as transcript) is subsequently beingtranslated into peptides, polypeptides, or proteins. The transcripts andthe encoded polypeptides are collectively referred to as gene product.If the polynucleotide is derived from genomic DNA, expression in aeukaryotic cell may include splicing of the mRNA.

A “vector” is a nucleic acid molecule, in particular self-replicating,which transfers an inserted nucleic acid molecule into and/or betweenhost cells. The term includes vectors that function primarily forinsertion of DNA or RNA into a cell (e.g., chromosomal integration),replication of vectors that function primarily for the replication ofDNA or RNA, and expression vectors that function for transcriptionand/or translation of the DNA or RNA. Also included are vectors thatprovide more than one of the functions as described.

An “expression vector” is a polynucleotide which, when introduced intoan appropriate host cell, can be transcribed and translated into apolypeptide. An “expression system” usually refers to a suitable hostcell comprised of an expression vector that can function to yield adesired expression product.

The following examples, sequence listing and figures are provided to aidthe understanding of the present invention, the true scope of which isset forth in the appended claims. It is understood that modificationscan be made in the procedures set forth without departing from thespirit of the invention.

Description of the Sequence Listing (Amino Acid Sequences)

-   SEQ ID NO:1 variable heavy chain domain VH of <VEGF> bevacizumab-   SEQ ID NO:2 variable light chain domain VL of <VEGF> bevacizumab-   SEQ ID NO:3 variable heavy chain domain VH of <ANG-2> E6Q-   SEQ ID NO:4 variable light chain domain VL of <ANG-2> E6Q-   SEQ ID NO: 5 XMab1-<VEGF> light chain-   SEQ ID NO: 6 XMab1-<ANG2> light chain-   SEQ ID NO: 7 XMab1-<VEGF> heavy chain-   SEQ ID NO: 8 XMab1-<ANG2> heavy chain-   SEQ ID NO: 9 XMab2-<VEGF> light chain-   SEQ ID NO: 10 XMab2-<ANG2> light chain-   SEQ ID NO: 11 XMab2-<VEGF> heavy chain-   SEQ ID NO: 12 XMab2-<ANG2> heavy chain-   SEQ ID NO: 13 XMab3-<VEGF> light chain-   SEQ ID NO: 14 XMab3-<ANG2> light chain-   SEQ ID NO: 15 XMab3-<VEGF> heavy chain-   SEQ ID NO: 16 XMab3-<ANG2> heavy chain-   SEQ ID NO: 17 XMab4-<VEGF> light chain-   SEQ ID NO: 18 XMab4-<ANG2> light chain-   SEQ ID NO: 19 XMab4-<VEGF> heavy chain-   SEQ ID NO: 20 XMab4-<ANG2> heavy chain-   SEQ ID NO: 21 XMab5-<VEGF> light chain-   SEQ ID NO: 22 XMab5-<ANG2> light chain-   SEQ ID NO: 23 XMab5-<VEGF> heavy chain-   SEQ ID NO: 24 XMab5-<ANG2> heavy chain-   SEQ ID NO: 25 XMab6-<VEGF> light chain-   SEQ ID NO: 26 XMab6-<ANG2> light chain-   SEQ ID NO: 27 XMab6-<VEGF> heavy chain-   SEQ ID NO: 28 XMab6-<ANG2> heavy chain-   SEQ ID NO: 29 OAscFab1-<ANG2> peptide connected heavy chain and    light chain-   SEQ ID NO: 30 OAscFab1-<VEGF> heavy chain-   SEQ ID NO: 31 OAscFab1-<VEGF> light chain-   SEQ ID NO: 32 OAscFab2-<ANG2> peptide connected heavy chain and    light chain-   SEQ ID NO: 33 OAscFab2-<VEGF> heavy chain-   SEQ ID NO: 34 OAscFab2-<VEGF> light chain-   SEQ ID NO: 35 OAscFab3-<ANG2> peptide connected heavy chain and    light chain-   SEQ ID NO: 36 OAscFab3-<VEGF> heavy chain-   SEQ ID NO: 37 OAscFab3-<VEGF> light chain-   SEQ ID NO:38 OAscXFab1-<ANG2> peptide connected heavy chain and    light chain-   SEQ ID NO: 39 OAscXFab1-<VEGF> heavy chain-   SEQ ID NO: 40 OAscXFab1-<VEGF> light chain-   SEQ ID NO: 41 OAscXFab2-<ANG2> peptide connected heavy chain and    light chain-   SEQ ID NO: 42 OAscXFab2-<VEGF> heavy chain-   SEQ ID NO: 43 OAscXFab2-<VEGF> light chain-   SEQ ID NO: 44 OAscXFab3-<ANG2> peptide connected heavy chain and    light chain-   SEQ ID NO: 45 OAscXFab3-<VEGF> heavy chain-   SEQ ID NO: 46 OAscXFab3-<VEGF> light chain-   SEQ ID NO: 47 Human vascular endothelial growth factor (VEGF)-   SEQ ID NO: 48 Human angiopoietin-2 (ANG-2)

EXPERIMENTAL PROCEDURES Examples Materials & General Methods

General information regarding the nucleotide sequences of humanimmunoglobulins light and heavy chains is given in: Kabat, E. A., etal., Sequences of Proteins of Immunological Interest, 5th ed., PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991).Amino acids of antibody chains are numbered and referred to according toEU numbering (Edelman, G. M., et al., Proc. Natl. Acad. Sci. USA 63(1969) 78-85; Kabat, E. A., et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md., (1991)).

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook,J. et al., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions.

Gene Synthesis

Desired gene segments can be prepared from oligonucleotides made bychemical synthesis. The gene segments, which are flanked by singularrestriction endonuclease cleavage sites, were assembled by annealing andligation of oligonucleotides including PCR amplification andsubsequently cloned via the indicated restriction sites e.g. KpnI/SacIor AscI/PacI into a pPCRScript (Stratagene) based pGA4 cloning vector.The DNA sequences of the subcloned gene fragments were confirmed by DNAsequencing.

Gene synthesis fragments were ordered according to given specificationsat Geneart (Regensburg, Germany). All gene segments encoding light andheavy chains of Ang-2/VEGF bispecific antibodies were synthesized with a5′-end DNA sequence coding for a leader peptide (MGWSCIILFLVATATGVHS),which targets proteins for secretion in eukaryotic cells, and uniquerestriction sites at the 5′ and 3′ ends of the synthesized gene. DNAsequences carrying disulfide stabilized “knobs-into-hole” modified heavychains were designed with S354C and T366W mutations in the “knobs” heavychain and Y349C, T366S, L368A and Y407V mutations in the “hole” heavychain.

DNA Sequence Determination

DNA sequences were determined by double strand sequencing performed atMediGenomix GmbH (Martinsried, Germany) or Sequiserve GmbH(Vaterstetten, Germany).

DNA and Protein Sequence Analysis and Sequence Data Management

The GCG's (Genetics Computer Group, Madison, Wis.) software packageversion 10.2 and Infomax's Vector NT1 Advance suite version 8.0 was usedfor sequence creation, mapping, analysis, annotation and illustration.

Expression Vectors

For the expression of the described antibodies variants of expressionplasmids for transient expression (e.g. in HEK293 EBNA or HEK293-F)cells or for stable expression (e.g. in CHO cells) based either on acDNA organization with a CMV-Intron A promoter or on a genomicorganization with a CMV promoter (e.g. FIG. 2B) were applied.

Beside the antibody expression cassette the vectors contained:

-   -   an origin of replication which allows replication of this        plasmid in E. coli, and    -   a β-lactamase gene which confers ampicillin resistance in E.        coli.

The transcription unit of the antibody gene is composed of the followingelements:

-   -   unique restriction site(s) at the 5′ end    -   the immediate early enhancer and promoter from the human        cytomegalovirus,    -   followed by the Intron A sequence in the case of the cDNA        organization,    -   a 5′-untranslated region of a human antibody gene,    -   a immunoglobulin heavy chain signal sequence,    -   the human antibody chain (heavy chain, modified heavy chain or        light chain) either as cDNA or as genomic organization with an        the immunoglobulin exon-intron organization    -   a 3′ untranslated region with a polyadenylation signal sequence,        and    -   unique restriction site(s) at the 3′ end.

For transient and stable transfections larger quantities of the plasmidswere prepared by plasmid preparation from transformed E. coli cultures(Nucleobond AX, Macherey-Nagel).

Cell Culture Techniques

Standard cell culture techniques were used as described in CurrentProtocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford,J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley &Sons, Inc.

Transient Transfections in HEK293-F System

Recombinant immunoglobulin variants were expressed by transienttransfection of human embryonic kidney 293-F cells using the FreeStyle™293 Expression System according to the manufacturer's instruction(Invitrogen, USA). Briefly, suspension FreeStyle™ 293-F cells werecultivated in FreeStyle™ 293 Expression medium at 37° C./8% CO₂ and thecells were seeded in fresh medium at a density of 1-2×10⁶ viablecells/ml on the day of transfection. DNA-293Fectin™ complexes wereprepared in Opti-MEM® I medium (Invitrogen, USA) using 325 μl of293Fectin™ (Invitrogen, Germany) and 250 μg of heavy and light chainplasmid DNA in a 1:1 molar ratio for a 250 ml final transfection volumefor monospecific parent antibodies. “Knobs-into-hole” DNA-293fectincomplexes with two heavy chains and one light chain were prepared inOpti-MEM® I medium (Invitrogen, USA) using 325 μl of 293Fectin™(Invitrogen, Germany) and 250 μg of “Knobs-into-hole” heavy chain 1 and2 and light chain plasmid DNA generally in a 1:1:1 molar ratio for a 250ml final transfection volume (OAscFab and OAscXFab). For expressionyield optimization the ratio can be varied. XMab DNA-293fectin complexeswere prepared in Opti-MEM® I medium (Invitrogen, USA) using 325 μl of293Fectin™ (Invitrogen, Germany) and 250 μg of “Knobs-into-hole” heavychain 1 and 2 and light chain plasmid DNA in a 1:1:1:1 molar ratio for a250 ml final transfection volume. For expression yield optimization theratio can be varied. Antibody containing cell culture supernatants wereharvested 7 days after transfection by centrifugation at 14000 g for 30minutes and filtered through a sterile filter (0.22 μm). Supernatantswere stored at −20° C. until purification.

Protein Determination

The protein concentration of purified antibodies and derivatives wasdetermined by determining the optical density (OD) at 280 nm, using themolar extinction coefficient calculated on the basis of the amino acidsequence according to Pace, C. N., et. al., Protein Science 4 (1995)2411-1423.

Antibody Concentration Determination in Supernatants

The concentration of antibodies and derivatives in cell culturesupernatants was estimated by immunoprecipitation with Protein AAgarose-beads (Roche). 60 μL Protein A Agarose beads are washed threetimes in TBS-NP40 (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet-P40).Subsequently, 1-15 mL cell culture supernatant are applied to theProtein A Agarose beads pre-equilibrated in TBS-NP40. After incubationfor at 1 h at room temperature the beads are washed on anUltrafree-MC-filter column (Amicon] once with 0.5 mL TBS-NP40, twicewith 0.5 mL 2× phosphate buffered saline (2×PBS, Roche) and briefly fourtimes with 0.5 mL 100 mM Na-citrate pH 5.0. Bound antibody is eluted byaddition of 35 μl NuPAGE® LDS Sample Buffer (Invitrogen). Half of thesample is combined with NuPAGE® Sample Reducing Agent or left unreduced,respectively, and heated for 10 min at 70° C. Consequently, 20 μl areapplied to an 4-12% NuPAGE® Bis-Tris SDS-PAGE (Invitrogen) (with MOPSbuffer for non-reduced SDS-PAGE and MES buffer with NuPAGE® Antioxidantrunning buffer additive (Invitrogen) for reduced SDS-PAGE) and stainedwith Coomassie Blue.

The concentration of antibodies and derivatives in cell culturesupernatants was measured by Protein A-HPLC chromatography. Briefly,cell culture supernatants containing antibodies and derivatives thatbind to Protein A were applied to a HiTrap Protein A column (GEHealthcare) in 50 mM K2HPO4, 300 mM NaCl, pH 7.3 and eluted from thematrix with 550 mM acetic acid, pH 2.5 on a Dionex HPLC-System. Theeluted protein was quantified by UV absorbance and integration of peakareas. A purified standard IgG1 antibody served as a standard.

Alternatively, the concentration of antibodies and derivatives in cellculture supernatants was measured by Sandwich-IgG-ELISA. Briefly,StreptaWell High Bind Strepatavidin A-96 well microtiter plates (Roche)were coated with 100 μL/well biotinylated anti-human IgG capturemolecule F(ab′)2<h-Fcgamma> BI (Dianova) at 0.1 μg/mL for 1 h at roomtemperature or alternatively over night at 4° C. and subsequently washedthree times with 200 μL/well PBS, 0.05% Tween (PBST, Sigma). 100 μL/wellof a dilution series in PBS (Sigma) of the respective antibodycontaining cell culture supernatants was added to the wells andincubated for 1-2 h on a microtiterplate shaker at room temperature. Thewells were washed three times with 200 μL/well PBST and bound antibodywas detected with 100 μl F(ab′)2<hFcgamma>POD (Dianova) at 0.1 μg/mL asdetection antibody for 1-2 h on a microtiterplate shaker at roomtemperature. Unbound detection antibody was washed away three times with200 μL/well PBST and the bound detection antibody was detected byaddition of 100 μL ABTS/well. Determination of absorbance was performedon a Tecan Fluor Spectrometer at a measurement wavelength of 405 nm(reference wavelength 492 nm).

Purification of Bispecific Antibodies

Bispecific antibodies were purified from cell culture supernatants byaffinity chromatography using Protein A-Sepharose™ (GE Healthcare,Sweden) and Superdex200 size exclusion chromatography. Briefly, sterilefiltered cell culture supernatants were applied on a HiTrap ProteinA HP(5 ml) column equilibrated with PBS buffer (10 mM Na₂HPO₄, 1 mM KH₂PO₄,137 mM NaCl and 2.7 mM KCl, pH 7.4). Unbound proteins were washed outwith equilibration buffer. Antibody and antibody variants were elutedwith 0.1 M citrate buffer, pH 2.8, and the protein containing fractionswere neutralized with 0.1 ml 1 M Tris, pH 8.5. Then, the eluted proteinfractions were pooled, concentrated with an Amicon Ultra centrifugalfilter device (MWCO: 30 K, Millipore) to a volume of 3 ml and loaded ona Superdex200 HiLoad 120 ml 16/60 gel filtration column (GE Healthcare,Sweden) equilibrated with 20 mM Histidin, 140 mM NaCl, pH 6.0. Fractionscontaining purified bispecific antibodies with less than 5% highmolecular weight aggregates were pooled and stored as 1.0 mg/ml aliquotsat −80° C.

SDS-PAGE

The NuPAGE® Pre-Cast gel system (Invitrogen) was used according to themanufacturer's instruction. In particular, 4-20% NuPAGE® Novex®TRIS-Glycine Pre-Cast gels and a Novex® TRIS-Glycine SDS running bufferwere used. (see e.g. FIG. 3). Reducing of samples was achieved by addingNuPAGE® sample reducing agent prior to running the gel.

Analytical Size Exclusion Chromatography

Size exclusion chromatography for the determination of the aggregationand oligomeric state of antibodies was performed by HPLC chromatography.Briefly, Protein A purified antibodies were applied to a Tosoh TSKgelG3000SW column in 300 mM NaCl, 50 mM KH2PO4/K2HPO4, pH 7.5 on an AgilentHPLC 1100 system or to a Superdex 200 column (GE Healthcare) in 2×PBS ona Dionex HPLC-System. The eluted protein was quantified by UV absorbanceand integration of peak areas. BioRad Gel Filtration Standard 151-1901served as a standard. (see e.g. FIG. 4).

Mass Spectrometry

The total deglycosylated mass of crossover antibodies was determined andconfirmed via electrospray ionization mass spectrometry (ESI-MS).Briefly, 100 μg purified antibodies were deglycosylated with 50 mUN-Glycosidase F (PNGaseF, ProZyme) in 100 mM KH2PO4/K2HPO4, pH 7 at 37°C. for 12-24 h at a protein concentration of up to 2 mg/ml andsubsequently desalted via HPLC on a Sephadex G25 column (GE Healthcare).The mass of the respective heavy and light chains was determined byESI-MS after deglycosylation and reduction. In brief, 50 μg antibody in115 μl were incubated with 60 μl 1M TCEP and 50 μl 8 MGuanidine-hydrochloride subsequently desalted. The total mass and themass of the reduced heavy and light chains was determined via ESI-MS ona Q-Star Elite MS system equipped with a NanoMate source.

Generation of HEK293-Tie2 Cell Line

In order to determine the interference of Angiopoietin-2 antibodies withANGPT2 stimulated Tie2 phosphorylation and binding of ANGPT2 to Tie2 oncells a recombinant HEK293-Tie cell line was generated. Briefly, apcDNA3 based plasmid (RB22-pcDNA3 Topo hTie2) coding for full-lengthhuman Tie2 (SEQ ID 108) under control of a CMV promoter and a Neomycinresistance marker was transfected using Fugene (Roche Applied Science)as transfection reagent into HEK293 cells (ATCC) and resistant cellswere selected in DMEM 10% FCS, 500 μg/ml G418. Individual clones wereisolated via a cloning cylinder, and subsequently analyzed for Tie2expression by FACS. Clone 22 was identified as clone with high andstable Tie2 expression even in the absence of G418 (HEK293-Tie2clone22). HEK293-Tie2 clone22 was subsequently used for cellular assays:ANGPT2 induced Tie2 phosphorylation and ANGPT2 cellular ligand bindingassay.

ANGPT2 Induced Tie2 Phosphorylation Assay

Inhibition of ANGPT2 induced Tie2 phosphorylation by ANGPT2 antibodieswas measured according to the following assay principle. HEK293-Tie2clone22 was stimulated with ANGPT2 for 5 minutes in the absence orpresence of ANGPT2 antibody and P-Tie2 was quantified by a sandwichELISA. Briefly, 2×105 HEK293-Tie2 clone 22 cells per well were grownover night on a Poly-D-Lysine coated 96 well-microtiter plate in 100 μlDMEM, 10% FCS, 500 μg/ml Geneticin. The next day a titration row ofANGPT2 antibodies was prepared in a microtiter plate (4-foldconcentrated, 75 μl final volume/well, duplicates) and mixed with 75 μlof an ANGPT2 (R&D systems #623-AN] dilution (3.2 μg/ml as 4-foldconcentrated solution). Antibodies and ANGPT2 were pre-incubated for 15min at room temperature. 100 μl of the mix were added to the HEK293-Tie2clone 22 cells (pre-incubated for 5 min with 1 mM NaV3O4, Sigma #S6508)and incubated for 5 min at 37° C. Subsequently, cells were washed with200 μl ice-cold PBS+1 mM NaV3O4 per well and lysed by addition of 120 μllysis buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 1% NP-40, 10% glycerol, 2mM EDTA, 1 mM NaV3O4, 1 mM PMSF and 10 μg/ml Aprotinin) per well on ice.Cells were lysed for 30 min at 4° C. on a microtiter plate shaker and100 μl lysate were transferred directly into a p-Tie2 ELISA microtiterplate (R&D Systems, R&D #DY990) without previous centrifugation andwithout total protein determination. P-Tie2 amounts were quantifiedaccording to the manufacturer's instructions and IC50 values forinhibition were determined using XLfit4 analysis plug-in for Excel(Dose-response one site, model 205). IC50 values can be compared withinon experiment but might vary from experiment to experiment.

VEGF Induced HUVEC Proliferation Assay

VEGF induced HUVEC (Human Umbilical Vein Endothelial Cells, Promocell#C-12200) proliferation was chosen to measure the cellular function ofVEGF antibodies. Briefly, 5000 HUVEC cells (low passage number, ≦5passages) per 96 well were incubated in 100 μl starvation medium (EBM-2Endothelial basal medium 2, Promocell # C-22211, 0.5% FCS,Penicilline/Streptomycine) in a collagen I-coated BD Biocoat Collagen I96-well microtiter plate (BD #354407/35640 over night. Varyingconcentrations of antibody were mixed with rhVEGF (30 ngl/ml finalconcentration, BD #354107) and pre-incubated for 15 minutes at roomtemperature. Subsequently, the mix was added to the HUVEC cells and theywere incubated for 72 h at 37° C., 5% CO2. On the day of analysis theplate was equilibrated to room temperature for 30 min and cellviability/proliferation was determined using the CellTiter-Glo™Luminescent Cell Viability Assay kit according to the manual (Promega, #G7571/2/3). Luminescence was determined in a spectrophotometer.

Example 1a Expression & Purification of Bispecific, Bivalent DomainExchanged <VEGF-ANG-2> Antibody Molecules XMab

According the procedures described in the materials and methods above,the bispecific, bivalent domain exchanged <VEGF-ANG-2> antibodymolecules XMab1, XMab1 and XMab3 were expressed and purified. The VH andVL of <VEGF> part (SEQ ID NO:1 and SEQ ID NO:2) are based onbevacizumab. The VH of <ANG2> part (SEQ ID NO:3) was derived by a E6Qmutation (the original amino acid glutamic acid (E) at position 6 wasreplaced by glutamine (Q)) of the VH sequence of ANG2i-LC06 (which isdescribed in the PCT application No. PCT/EP2009/007182(WO2010/040508)—and which is further maturated fragment of a sequenceobtained via phage display). The VL of <ANG2> part (SEQ ID NO:4) wasderived from the VL sequences ANG2i-LC06 (see PCT application No.PCT/EP2009/007182 (WO2010/040508)). The bispecific, bivalent domainexchanged <VEGF-ANG-2> antibody molecules XMab1, XMab2 and XMab3 wereexpressed and purified. The relevant light and heavy chain amino acidsequences of these bispecific, bivalent antibodies are given in SEQ IDNO: 5-8 (XMab1), in SEQ ID NO: 9-12 (XMab2), and in SEQ ID NO: 13-16(XMab3). For an exemplary structure see FIG. 1.

Key data XMab1 XMab2 XMab3 Expression (Yield) 32 μg/mL 10 μg/mL 39 μg/mLPurification (Yield, Prot. A. 31 mg/L, 64% 8 μg/mL, 80% — homog.)

The bispecific, bivalent <VEGF-ANG-2> antibodies XMab4, XMab5 and XMab6(with the relevant light and heavy chain amino acid sequences given inSEQ ID NO: 17-20 (XMab4), in SEQ ID NO: 21-24 (XMab5), and in SEQ ID NO:25-28 (XMab6)) are expressed and purified analogously.

Binding affinities and other properties were or are determined asdescribed.

Example 1b Expression & Purification Bispecific, Bivalent <VEGF-ANG-2>Antibody Molecules OAscFab

According to the procedures described in the materials and methodsabove, the bispecific, bivalent <VEGF-ANG-2> antibody moleculesOAscFab1, OAscFab2, OAscFab3 were expressed and purified. The VH and VLof <VEGF> part (SEQ ID NO:1 and SEQ ID NO:2) are based on bevacizumab.The VH of <ANG2>E6Q part (SEQ ID NO:3) was derived by a E6Q mutation(the original amino acid glutamic acid (E) at position 6 was replaced byglutamine (Q)) of the VH sequences ANG2i-LC06 (which is described in thePCT application No. PCT/EP2009/007182 (WO2010/040508)—and which isfurther maturated fragment of a sequence obtained via phage display).The VL of <ANG2>E6Q part (SEQ ID NO:4) was derived from the VL sequenceof ANG2i-LC06 (see PCT application No. PCT/EP2009/007182 (WO2010/040508)(WO2010/040508)). The relevant light and heavy chain amino acidsequences of these bispecific, bivalent antibodies are given in SEQ IDNO: 29-31 (OAscFab1), in SEQ ID NO: 32-34 (OAscFab2), and in SEQ ID NO:35-37 (OAscFab3). For an exemplary structure see FIG. 2 a. Expression ofOAscFab1, OAscFab1, OAscFab2 and OAscFab3 was confirmed by Western blot.Purification of OAscFab2 and OAscFab3 led to the following yields.

Protein A SEC Antibody Supernatant Yield Mono. Yield Monomer OAscFab20.5 L 36.0 mg 86% 21.7 mg >95% OAscFab3 0.5 L 29.3 mg 85% 17.7 mg >95%

Binding affinities and other properties are determined as described.

Example 1c Expression & Purification Bispecific, Bivalent DomainExchanged <VEGF-ANG-2> Antibody Molecules OAscXFab

According the procedures described in the materials and methods above,the bispecific, bivalent domain exchanged <VEGF-ANG-2> antibodymolecules OAscXFab1, OAscXFab2, OAscXFab3, were expressed and purified.The VH and VL of <VEGF> part (SEQ ID NO:1 and SEQ ID NO:2) are based onbevacizumab. The VH of <ANG2>E6Q part (SEQ ID NO:3) was derived by a E6Qmutation (the original amino acid glutamic acid (E) at position 6 wasreplaced by glutamine (Q)) of the VH sequences ANG2i-LC06 (which isdescribed in the PCT application No. PCT/EP2009/007182(WO2010/040508)—and which is further maturated fragment of a sequenceobtained via phage display). The VL of <ANG2>E6Q part (SEQ ID NO:4) wasderived from the VL sequence of ANG2i-LC06 (see PCT application No.PCT/EP2009/007182 (WO2010/040508)). The relevant light and heavy chainamino acid sequences of these bispecific, bivalent antibodies are givenin SEQ ID NO: 38-40 (OAscXFab1), in SEQ ID NO: 41-43 (OAscXFab2), and inSEQ ID NO: 44-46 (OAscXFab3). For an exemplary structure see FIG. 2 b(OAscXFab1) and FIG. 2 c (OAscXFab2, OAscXFab3). Expression wasconfirmed by Western blot.

Key data OAscXFab1 OAscXFab2 OAscXFab3 Expression (Yield) 23 μg/mL 23μg/mL 26 μg/mL

Binding affinities and other properties are determined as described.

Example 2 Stability of Bispecific Antibodies Denaturation Temperature(SYPRO Orange Method)

To determine the temperature at which protein denaturation (i.e.temperature-induced loss of protein structure) occurs, a method was usedthat relies a hydrophobic fluorescent dye (SYPRO orange, Invitrogen)that exhibits strong fluorescence in hydrophobic environments. Uponprotein denaturation, hydrophobic patches become exposed to the solvent,leading to an increased fluorescence. At temperatures above thedenaturation temperature, fluorescence intensities decrease again, hencethe temperature at which a maximum intensity is reached is defined asthe denaturation temperature. The method is described by Ericsson, U.B., et al., Anal Biochem 357 (2006) 289-298 and He, F., et al., Journalof Pharmaceutical Sciences 99 (2010) 1707-1720.

Proteins samples at a concentration of approx. 1 mg/mL in 20 mMHis/HisCl, 140 mM NaCl, pH 6.0 were mixed with SYPRO orange (5000× stocksolution) to reach a final dilution of 1:5000. A volume of 20 μL wastransferred into a 384 well-plate and temperature-dependent fluorescencewas recorded in a LightCycler® 480 Real-Time PCR System (Roche AppliedSciences) at a heat rate of 0.36° C./min.

Aggregation Temperature by Dynamic Light Scattering (DLS)

The temperature at which thermally induced protein aggregation occurswas determined by dynamic light scattering (DLS). DLS yields informationon the size distribution of macromolecules in solution, derived fromfluctuations of scattered light intensities on a microsecond scale. Whensamples are heated up gradually, aggregation starts at a certaintemperature, giving rise to growing particle sizes. The temperature atwhich particle sizes begin to increase is defined as the aggregationtemperature. Aggregation and denaturation temperatures need notnecessarily be identical since denaturation may not necessarily be aprerequisite for aggregation.

For aggregation temperature measurements, a DynaPro DLS platereader(Wyatt technologies) was used. Preceding the measurement, samples werefiltered via 384-well filter plates (Millipore MultiScreen 384-wellFiltration System, 0.45 μm) into optical 384 well plates (Corning#3540). A sample volume of 35 μL was used at a protein concentration ofapprox. 1 mg/mL in formulation buffer (20 mM citrate, 180 mM sucrose, 20mM arginine, 0.02% polysorbate 20). Each well was covered with 20 μLparaffin oil (Sigma) to avoid evaporation. Samples were heated from 25°C. to 80° C. at a rate of 0.05° C./min and DLS data were acquiredcontinuously for a maximum number of 15 samples per run.

Aggregation Rate Per DLS

DLS is a sensitive method for detecting aggregates of macromolecules insolution, since aggregates give rise to strong light scattering signals.Hence, the tendency of a molecule to aggregate can be followed over timeby repeated acquisition of DLS data. To accelerate potential aggregationto practical rates, measurements were conducted at 50° C.

Sample preparation was performed as described above. DLS data wererecorded for up to 100 hours. Aggregation rates (nm/day) were calculatedas the slope of a linear fit of average diameters over time.

Stability in Formulation Buffer

To assess bispecific molecules for their stability with regard toaggregation/fragmentation, samples were incubated for 3 weeks at 40° C.in formulation buffer (20 mM citrate, 180 mM sucrose, 20 mM arginine,0.02% polysorbate 20) at a protein concentration of approximately 1mg/mL. A control sample was stored for 3 weeks at −80° C.

Size exclusion chromatography for the quantification of aggregates andlow-molecular weight (LMW) species was performed by HPLC. An amount of25-100 μg of protein was applied to a Tosoh TSKgel G3000SWXL column in300 mM NaCl, 50 mM potassium phosphate, pH 7.5 on an Ultimate3000 HPLCsystem (Dionex). The eluted protein was quantified by UV absorbance at280 nm.

Results

Stability of XMab1 Method (SEQ ID NO: 5-8) Denaturation temperature(SYPRO orange 71° C. method) Aggregation temperature by Dynamic 65° C.Light Scattering (DLS) Aggregation rate per DLS 0.04 nm/day Stability informulation buffer ΔHMW: 0.6 area % (difference between 40° C. and −80°C. after 3 ΔLMW: 0.5 area % weeks storage) ΔMonomer: −1.2 area %

Example 3 Binding Properties of Bispecific Antibody <VEGF-Ang-2>

A) Binding properties characterized by Surface Plasmon Resonance (SPR)Analysis

Simultaneous binding of both antigens was confirmed by applying SurfacePlasmon Resonance (SPR) using a BIAcore T100 instrument (GE HealthcareBiosciences AB, Uppsala, Sweden). VEGF was immobilized to a CM5Sensorchip using standard amine coupling chemistry. In a first step,<VEGF-Ang-2> XMAb was injected at a concentration of 10 μg/ml in HBSbuffer (10 mM HEPES, 150 mM NaCl, 0.05% Tween 20, pH 7.4) at 25° C.After binding of the antibody to the immobilized VEGF, hAng-2 wasinjected at 10 μg/ml in a second step (FIG. 3)

In a further experiment the affinity and binding kinetics of<VEGF-Ang-2> XMab were determined. Briefly, goat<hIgG-Fcgamma>polyclonal antibodies were immobilized on a CM4 chip via amine couplingfor presentation of the bispecific antibody against Ang-2 and VEGF.Binding was measured in HBS buffer at 25° C. or 37° C. PurifiedAng-2-His (R&D systems or in house purified) or VEGF (R&D systems or inhouse purified) was added in various concentrations between 0.37 nM and30 nM or between 3.7 nM and 200 nM in solution. Association was measuredby an injection of 3 minutes; dissociation was measured by washing thechip surface with HBS buffer for 10 minutes and a KD value was estimatedusing a 1:1 Langmuir binding model. Due to heterogenity of the Ang-2preparation no 1:1 binding could be observed. Therefore KD values areapparent values. The determined affinity of <VEGF-Ang-2> XMab to VEGFwas extremely high, the calculated off-rate was out of Biacorespecifications even at 37° C. In Table 1 the binding constants for bothantigens are summarized.

TABLE 1 Kinetic parameters of <VEGF-Ang-2> XMab1 binding to Ang-2 andVEGF Apparent ka Apparent kd Apparent KD Analyte (1/Ms) (1/s) (M) Ang-22.7E+06 6.3E−04 2.4E−10 VEGF 1.2E+05  <1E−06  <1E−10

B) Assay for Quantification of Binding Active Bispecific <Ang2/VEGF>XMab1

Additionally to the SPR analysis, an ELISA was established to quantifythe amount of binding active bispecific mAb<Ang2/VEGF> antibodies. Inthis assay, hAng2 is directly coated to the wells of a maxisorpmicrotiter plate (MTP) in the first step. Meanwhile, thesamples/reference standards (mAb<Ang2/VEGF>) were pre-incubated in thewells of another MTP with digoxigenylated VEGF. After pre-incubation andcoating, excess of unbound Ang2 was removed by washing the Ang2 coatedMTP. The pre-incubated mixture of <Ang2/VEGF> and VEGF-Dig was thentransferred to the hAng2 coated MTP and incubated. After incubation, theexcess of pre-incubation solution was removed by washing followed byincubation with a horse-radish peroxidase labeled anti-digoxigeninantibody. The antibody-enzyme conjugate catalyzes the color reaction ofthe ABTS® substrate. The signal was measured by ELISA reader at 405 nmwavelength (reference wavelength: 490 nm ([405/490] nm)). Absorbancevalues of each sample were determined in duplicates. (A schemeexemplifying this test system is shown in FIG. 4 and Calibration curveof ELISA for quantification is shown in FIG. 5)

Example 4 Tie2 Phosphorylation

In order to confirm that the anti-ANGPT2 related activities are retainedin the bispecific bivalent <VEGF-ANGPT2> antibody XMAb1 Tie2phosphorylation assay was performed. The efficacy of XMAb1 wasdetermined in the ANGPT2 stimulated Tie2 phosphorylation assay asdescribed above.

It was shown that XMAb1 interferes with ANGPT2 stimulated Tie2phosphorylation in the ANGPT2 stimulated Tie2 phosphorylation assay asdescribed above. IC50 for XMAb1 was 7.4 nM+/−2.3.

Example 5 Inhibition of huANG-2 Binding to Tie-2 (ELISA)

The interaction ELISA was performed on 384 well microtiter plates(MicroCoat, DE, Cat. No. 464718) at RT. After each incubation stepplates were washed 3 times with PBST. ELISA plates were coated with 5μg/ml Tie-2 protein for 1 hour (h). Thereafter the wells were blockedwith PBS supplemented with 0.2% Tween-20 and 2% BSA (Roche DiagnosticsGmbH, DE) for 1 h. Dilutions of purified bispecific Xmab antibodies inPBS were incubated together with 0.2 μg/ml huAngiopoietin-2 (R&DSystems, UK, Cat. No. 623-AN) for 1 h at RT. After washing a mixture of0.5 μg/ml biotinylated anti-Angiopoietin-2 clone BAM0981 (R&D Systems,UK) and 1:3000 diluted streptavidin HRP (Roche Diagnostics GmbH, DE,Cat. No. 11089153001) was added for 1 h. Thereafter the plates werewashed 3 times with PBST. Plates are developed with freshly preparedABTS reagent (Roche Diagnostics GmbH, DE, buffer #204 530 001, tablets#11 112 422 001) for 30 minutes at RT. Absorbance was measured at 405 nmand the 1050 was determined. XMab1 showed an inhibition of ANG-2 bindingto Tie-2 with an IC50 of 12 nM.

Example 6 Inhibition of hVEGF Binding to hVEGF Receptor (ELISA)

The test was performed on 384 well microtiter plates (MicroCoat, DE,Cat. No. 464718) at RT. After each incubation step plates were washed 3times with PBST. At the beginning, plates were coated with 1 μg/mlhVEGF-R protein (R&D Systems, UK, Cat. No. 321-FL) for 1 hour (h).Thereafter the wells were blocked with PBS supplemented with 0.2%Tween-20 and 2% BSA (Roche Diagnostics GmbH, DE) for 1 h. Dilutions ofpurified bispecific XMab antibodies in PBS were incubated together with0.15 μg/ml huVEGF121 (R&D Systems, UK, Cat. No. 298-VS) for 1 h at RT.After washing a mixture of 0.5 μg/ml anti VEGF clone Mab923 (R&DSystems, UK) and 1:2000 horse radish peroxidase (HRP)-conjugated F(ab′)2anti mouse IgG (GE Healthcare, UK, Cat. No. NA9310V) was added for 1 h.Thereafter the plates were washed 6 times with PBST. Plates weredeveloped with freshly prepared ABTS reagent (Roche Diagnostics GmbH,DE, buffer #204 530 001, tablets #11 112 422 001) for 30 minutes at RT.Absorbance was measured at 405 nm and the 1050 was determined. XMab1showed an inhibition of Inhibition of VEGF binding to VEGF Receptor withan IC50 of 10 nM.

Example 7 HUVEC Proliferation

In order to confirm that the anti-VEGF related activities are retainedin the bispecific bivalent <VEGF-ANG2> antibody XMAb1 VEGF-induced HUVECproliferation assay was performed. It was shown that XMAb1 interfereswith VEGF-induced HUVEC proliferation in a comparable manner asbevacizumab in the VEGF-induced HUVEC proliferation assay as describedabove. XMAb1 interferes in a concentration dependent manner withVEGF-induced HUVEC proliferation comparable to the parental antibodybevacizumab (Avastin). IC50 was 1.1 nM for bevacizumab and 2.3 nM forXMAb.

Example 8 Mouse Cornea Micropocket Angiogenesis Assay

8 to 10 weeks old female Balb/c mice were purchased from Charles River,Sulzfeld, Germany. The protocol was modified according to the methoddescribed by Rogers, M. S., et al., Nat Protoc 2 (2007) 2545-2550.Briefly, micropockets with a width of about 500 μm were prepared under amicroscope at approximately 1 mm from the limbus to the top of thecornea using a surgical blade and sharp tweezers in the anesthetizedmouse. The disc (Nylaflo®, Pall Corporation, Michigan) with a diameterof 0.6 mm was implanted and the surface of the implantation area wassmoothened. Discs were incubated in corresponding growth factor or invehicle for at least 30 min. After 3, 5 and 7 days (or alternativelyonly after 3 days), eyes were photographed and vascular response wasmeasured. The assay was quantified by calculating the percentage of thearea of new vessels per total area of the cornea.

The discs were loaded with 300 ng VEGF or with PBS as a control andimplanted for 7 days. The outgrowth of vessels from the limbus to thedisc was monitored over time on day 3, 5 and 7. One day prior to discimplantation the antibodies (<Ang-2/VEGF> XMAb1, <hVEGF> Avastin(bevacizumab)) were administered intravenously at a dose of 10 mg/kg forAvastin and XMAb1. Animals in the control group received vehicle. Theapplication volume was 10 ml/kg.

To test the effect of XMAb1 on VEGF-induced angiogenesis in vivo, weperformed the mouse corneal angiogenesis assay. In this assay a VEGFsoaked Nylaflo disc is implanted into a pocket of the avascular corneaat a fixed distance to the limbal vessels. Vessels immediately grow intothe cornea towards the developing VEGF gradient. Our results demonstratethat systemic administration of the XMAb1 (10 mg/kg) almost completelyinhibited the outgrowth of the vessel from the limbus towards the VEGFgradient from study day 3 to 5 (FIG. 6). In a further experiment, directcomparison studies were performed. The discs were loaded with 300 ngVEGF or with PBS as a control and implanted for 3 days. The outgrowth ofvessels from the limbus to the disc was monitored over time on day 3.One day prior to disc implantation the antibodies (bispecific<Ang-2/VEGF> antibody XMAb1, parent <VEGF> antibody bevacizumab(Avastin), parent <Ang-2> antibody ANG2i-LC06, and the combination of<VEGF> antibody bevacizumab (Avastin) and <Ang-2> antibody ANG2i-LC06)were administered intravenously at a dose of 10 mg/kg for bevacizumab(Avastin), 10 mg/kg for XMAb1, 10 mg/kg for bevacizumab (Avastin), and10 mg/kg for ANG2i-LC06. The combination of bevacizumab (Avastin) andANG2i-LC06 was administered with 10 mg/kg for bevacizumab (Avastin) and10 mg/kg for ANG2i-LC06. Animals in the control group received vehicle.The application volume was 10 ml/kg.

Our results (see FIG. 7 and Table below) demonstrate that systemicadministration of the XMAb1 (10 mg/kg) almost completely inhibited theoutgrowth of the vessel from the limbus towards the VEGF gradient atstudy day 3 comparable to the combination of bevacizumab and ANG2i-LC06.Anti-Ang-2 monotherapy in contrast only slightly inhibited VEGF-inducedangiogenesis (FIG. 7). The maximum effect could already be achieved atlower concentrations of 10 mg/kg XMAb1 compared to the combination of 10mg/kg of ANG2i-LC06+10 mg/kg of bevacizumab (Avastin).

TABLE Percent inhibition of VEGF-induced angiogenesis on day 3 in aMouse cornea micropocket angiogenesis assay % inhibition of VEGF-inducedangiogenesis day 3 VEGF (300 ng) 0 ANG2i-LC06 38 (10 mg/kg) Bevacizumab(Avastin) 83 (10 mg/kg) XMab1 (10 mg/kg) 96 Ang2i-LC06 (10 mg/kg) + 95bevacizumab (Avastin) (10 mg/kg)

Example 9 In Vivo Efficacy of Bispecific Antibody <VEGF-ANG-2> Antibodyin Colo205 Xenograft Model in Scid Beige Mice Cell Lines and CultureConditions:

Colo205 human colorectal cancer cells (ATCC No. CCL-222). Tumor cellline were routinely cultured in RPMI 1640 medium (PAA, Laboratories,Austria) supplemented with 10% fetal bovine serum (PAA Laboratories,Austria) and 2 mM L-glutamine, at 37° C. in a water-saturated atmosphereat 5% CO₂. Passage 2-5 is used for transplantation.

Animals:

Female SCID beige mice; age 4-5 weeks at arrival (purchased from CharlesRiver Germany) were maintained under specific-pathogen-free conditionwith daily cycles of 12 h light/12 h darkness according to committedguidelines (GV-Solas; Felasa; TierschG). Experimental study protocol wasreviewed and approved by local government. After arrival animals weremaintained in the quarantine part of the animal facility for one week toget accustomed to new environment and for observation. Continuous healthmonitoring is carried out on regular basis. Diet food (Provimi Kliba3337) and water (acidified pH 2.5-3) were provided ad libitum. Age ofmice at start of the study is about 10 weeks.

Tumor Cell Injection:

At the day of injection, tumor cells were harvested (trypsin-EDTA) fromculture flasks (Greiner) and transferred into 50 ml culture medium,washed once and resuspended in PBS. After an additional washing stepwith PBS and filtration (cell strainer; Falcon 100 μm) the final celltiter was adjusted to 2.5×10⁷/ml. Tumor cell suspension was carefullymixed with transfer pipette to avoid cell aggregation. After this, cellsuspension was filled into a 1.0 ml tuberculin syringe (Braun Melsungen)using a wide needle (1.10×40 mm); for injection needle size is changed(0.45×25 mm) and for every injection a new needle was used. Anesthesiawas performed using a Stephens inhalation unit for small animals withpreincubation chamber (plexiglas), individual mouse nose-mask (silicon)and not flammable or explosive anesthesia compound Isoflurane(cp-pharma) in a closed circulation system. Two days before injectioncoat of the animals was shaved and for cell injection skin ofanaesthetized animals was carefully lifted up with an anatomic forcepsand 100 μl cell suspension (=2.5×10⁶ cells) was injected subcutaneouslyin the right flank of the animals.

Treatment of Animals

Treatment of animals started at day of randomization at a mean tumorvolume of ˜100 mm3, respectively. Mice were treated once weekly i.p.with the different compounds as indicated in following table.

No of Route/Mode of animals Compound Dose (mg/kg) administration 10Xolair 10 i.p. once weekly 10 <VEGF> Avastin 10 i.p. once weekly 10<ANG-2> Ang2i- 10 i.p. once weekly LC06 10 Ang2i-LC06 + 10 i.p. onceweekly Avastin 10 i.p. once weekly 10 XMAb1 10 i.p. once weekly

Monitoring:

Animals were controlled 2× per week for their health status. Bodyweights were documented 2× per week after cell injection. The tumordimensions were measured by caliper beginning on the staging day andsubsequently 2 times per week during the whole treatment period. Tumorvolume was calculated according to NCI protocol (Tumor weight=½ab²,where “a” and “b” are the long and the short diameters of the tumor,respectively). Termination criteria were the critical tumor mass (up to1.7 g or Ø>1.5 cm), body weight loss more than 20% from baseline, tumorulceration or poor general condition of the animals.

The results (see (FIG. 8) show that the bispecific bivalent <VEGF-ANG-2>antibody XMAb1 showed a higher tumor growth inhibition in xenografttumor model Colo205 in Scid beige mice compared to the treatment withmonospecific antibodies. The efficacy of the combination of ANG2i-LC06and bevacizumab showed comparable results to the XMAb1. Maximal efficacyof XMAb1 was already reached with 10 mg/kg.

In a second experiment the effect of XMAb1 on bigger tumors wasanalyzed.

Treatment of Animals Treatment of animals started at day ofrandomization at a mean tumor volume of ˜400 mm3, respectively. Micewere treated once weekly i.p. with the different compounds as indicatedin following table.

No of Route/Mode of animals Compound Dose (mg/kg) administration 10Xolair 10 i.p. once weekly 10 <VEGF> Avastin 10 i.p. once weekly 10<ANG-2> Ang2i- 10 i.p. once weekly LC06 10 Ang2i-LC06 + 10 i.p. onceweekly Avastin 10 i.p. once weekly 10 XMAb1 10 i.p. once weekly

Monitoring:

Animals were controlled 2× per week for their health status. Bodyweights were documented 2× per week after cell injection. The tumordimensions were measured by caliper beginning on the staging day andsubsequently 2 times per week during the whole treatment period. Tumorvolume is calculated according to NCI protocol (Tumor weight=½ab², where“a” and “b” are the long and the short diameters of the tumor,respectively). Termination criteria were the critical tumor mass (up to1.7 g or Ø>1.5 cm), body weight loss more than 20% from baseline, tumorulceration or poor general condition of the animals.

The results (see FIG. 9) show that the bispecific bivalent <VEGF-ANG-2>antibody XMAb1 showed a higher tumor growth inhibition in xenografttumor model Colo205 in Scid beige mice compared to the treatment withmonospecific antibodies which showed no efficacy in big tumors comparedto the control. The efficacy of the combination of ANG2i-LC06 andbevacizumab showed comparable results to the XMAb1. Maximal efficacy ofXMAb1 was already reached with 10 m g/kg.

Taken together the results demonstrate that independent of the tumorsize XMAb1 shows superior efficacy compared to the treatment withmonospecific antibodies.

Tumor stasis in these models could already be achieved at lowerconcentrations of 10 mg/kg XMAb1 compared to the combination of 10 mg/kgof ANG2i-LC06+10 mg/kg of Avastin.

Example 10 In Vivo Efficacy of Bispecific Antibody <VEGF-ANG-2> Antibodyin Orthotopic KPL-4 Xenograft Model in Scid Beige Mice

Tumor Cell Line

The human breast cancer cell line KPL-4 ((Kurebayashi, J., et al., Br.J. Cancer 79 (1999) 707-17)) has been established from the malignantpleural effusion of a breast cancer patient with an inflammatory skinmetastasis. Tumor cells were routinely cultured in DMEM medium (PANBiotech, Germany) supplemented with 10% fetal bovine serum (PAN Biotech,Germany) and 2 mM L-glutamine (PAN Biotech, Germany) at 37° C. in awater-saturated atmosphere at 5% CO₂. Culture passage was performed withtrypsin/EDTA 1× (PAN) splitting three times/week.

Mice

After arrival, female SCID beige mice (age 10-12 weeks; body weight18-20 g) Charles River, Sulzfeld, Germany) were maintained in thequarantine part of the AALAAC approved animal facility for one week toget them accustomed to the new environment and for observation.Continuous health monitoring was carried out. The mice were kept underSPF-conditions according to the international guidelines (GV-Solas;Felasa; TierschG) with daily cycles of 12 h light/12 h darkness. Dietfood (Kliba Provimi 3347) and water (filtered) were provided ad libitum.Experimental study protocol was reviewed and approved by the localgovernment (Regierung von Oberbayern; registration no.211.2531.2-22/2003).

Tumor Cell Injection

At the day of injection tumor cells were harvested (trypsin-EDTA) fromculture flasks (Greiner TriFlask) and transferred into 50 ml culturemedium, washed once and resuspended in PBS. After an additional washingstep with PBS and filtration (cell strainer; Falcon Ø 100 μm) the finalcell titer was adjusted to 1.5×10⁸/ml. Tumor cell suspension wascarefully mixed with transfer pipette to avoid cell aggregation.Anesthesia is performed using a Stephens inhalation unit for smallanimals with preincubation chamber (plexiglas), individual mousenose-mask (silicon) and not flammable or explosive anesthesia compoundIsoflurane (Pharmacia-Upjohn, Germany) in a closed circulation system.Two days before injection coat of the animals were shaved. For i.m.f.p.injection cells were injected orthotopically at a volume of 20 μl intothe right penultimate inguinal mammary fat pad of each anesthetizedmouse. For the orthotopic implantation, the cell suspension was injectedthrough the skin under the nipple using a using a Hamilton microlitersyringe and a 30G×½″ needle.

Treatment of Animals

Treatment of animals started at day of randomization at a mean tumorvolume of ˜80 mm3, respectively. Mice were treated once weekly i.p. withthe different compounds as indicated in following table.

No of Route/Mode of animals Compound Dose (mg/kg) administration 10Xolair 10 i.p. once weekly 10 <VEGF> Avastin 10 i.p. once weekly 10<ANG-2> Ang2i- 10 i.p. once weekly LC06 10 Ang2i-LC06 + 10 i.p. onceweekly Avastin 10 i.p. once weekly 10 XMAb1 10 i.p. once weekly

Monitoring of Tumor Growth

Animals were controlled 2× per week for their health status. Bodyweights were documented 2× per week after cell injection. The tumordimensions were measured by caliper on the staging day, at beginning oftreatment period 2 times per week. Tumor volume was calculated accordingto NCI protocol (B. Teicher; Anticancer drug development guide, HumanaPress, 1997, Chapter 5, page 92) (Tumor weight=½ab², where “a” and “b”are the long and the short diameters of the tumor, respectively).

Termination criteria were the critical tumor mass (up to 1.7 g or Ø>1.5cm), body weight loss more than 20% from baseline, tumor ulceration orpoor general condition of the animals.

The results (see FIG. 10) show that the bispecific bivalent <VEGF-ANG-2>antibody XMAb1 showed a higher tumor growth inhibition in xenografttumor model Colo205 in Scid beige mice compared to the treatment withmonospecific antibodies. The efficacy of the combination of ANG2i-LC06and bevacizumab showed comparable results to the XMAb1. Maximal efficacyof XMAb1 was already reached with 10 mg/kg.

In a second experiment the effect of XMAb1 on bigger tumors wasanalyzed.

Treatment of Animals

Treatment of animals started at day of randomization at a mean tumorvolume of ˜160 mm3, respectively. Mice were treated once weekly i.p.with the different compounds as indicated in following table.

No of Route/Mode of animals Compound Dose (mg/kg) administration 10Xolair 10 i.p. once weekly 10 <VEGF> Avastin 10 i.p. once weekly 10<ANG-2> Ang2i- 10 i.p. once weekly LC06 10 Ang2i-LC06 + 10 i.p. onceweekly Avastin 10 i.p. once weekly 10 XMAb1 10 i.p. once weekly

Monitoring:

Animals were controlled 2× per week for their health status. Bodyweights were documented 2× per week after cell injection. The tumordimensions were measured by caliper on the staging day, at beginning oftreatment period 2 times per week. Tumor volume was calculated accordingto NCI protocol (B. Teicher; Anticancer drug development guide, HumanaPress, 1997, Chapter 5, page 92) (Tumor weight=½ab², where “a” and “b”are the long and the short diameters of the tumor, respectively).

Termination criteria were the critical tumor mass (up to 1.7 g or Ø>1.5cm), body weight loss more than 20% from baseline, tumor ulceration orpoor general condition of the animals.

The results (see FIG. 11) show that the bispecific bivalent <VEGF-ANG-2>antibody XMAb1 showed a higher tumor growth inhibition in xenografttumor model Colo205 in Scid beige mice compared to the treatment withmonospecific antibodies. The efficacy of the combination of ANG2i-LC06and bevacizumab showed comparable results to the XMAb1. Maximal efficacyof XMAb1 was already reached with 10 mg/kg.

Taken together the results demonstrate that independent of the tumorsize XMAb1 shows superior efficacy compared to the treatment withmonospecific antibodies.

Tumor stasis in these models could be already achieved at lowerconcentrations of 10 mg/kg XMAb1 compared to the combination of 10 mg/kgof Ang2i-LC06+10 mg/kg of Avastin.

Example 11 Effect of Treatment with XMAb1 on Micro-Vessel Density ins.c. Colo205 Xenograft

Vascular density is assessed by counting all vessels of a tumor slide.Vessels were labeled with fluorescent anti-mouse CD34 antibody (cloneMEC14.7) on paraffin-embedded sections. Vessels were quantified andmicrovessel density is calculated as vessels per mm². All results wereexpressed as mean±SEM. To define significant differences of experimentalgroups, Dunnetts-Method was used. p<0.05 was considered as statisticallysignificant. The results show that total intratumoral MVD was decreasedin treated tumors. Treatment with ANG2i-LC06 reduced MVD by 29%,bevacizumab by </=0%, bevacizumab+ANG2i-LC06 by 15% and XMAb1 by 28%.

Example 12 In Vivo Efficacy of Bispecific Antibody <VEGF-ANG-2> Antibodyin s.c. N87 Xenograft Model in Scid Beige Mice

Tumor Cell Line

The human gastric cancer cell line N87 cancer cells (NCI-N87 (ATCC No.CRL 5822)). Tumor cells were routinely cultured in RPMI1640 supplementedwith 10% fetal bovine serum (PAN Biotech, Germany) and 2 mM L-glutamine(PAN Biotech, Germany) at 37° C. in a water-saturated atmosphere at 5%CO₂. Culture passage was performed with trypsin/EDTA 1× (PAN) splittingthree times/week.

Mice

After arrival, female SCID beige mice (age 10-12 weeks; body weight18-20 g) Charles River, Sulzfeld, Germany) were maintained in thequarantine part of the AALAAC approved animal facility for one week toget them accustomed to the new environment and for observation.Continuous health monitoring was carried out. The mice were kept underSPF-conditions according to the international guidelines (GV-Solas;Felasa; TierschG) with daily cycles of 12 h light/12 h darkness. Dietfood (Kliba Provimi 3347) and water (filtered) were provided ad libitum.Experimental study protocol was reviewed and approved by the localgovernment (Regierung von Oberbayern; registration no.211.2531.2-22/2003).

Tumor Cell Injection

At the day of cell injection, cells were harvested from culture flasks(Greiner T 75), transferred into 50 ml culture medium, washed once andresuspended in PBS. After an additional washing with PBS the cellconcentration was measured with a Vi-Cell™ (Cell Viability Analyzer,Beckman Coulter, Madison, Wis., U.S.A.). The tumor cell suspension (PBS)was mixed carefully (to reduce cell aggregation) and kept on ice. Thecell suspension was filled into a 1.0 ml syringe. For injection, aneedle size of 0.45×25 mm was used. To generate primary tumors, 5×10⁶N87 tumor cells in a volume of 100 μl PBS were injected subcutaneouslyinto the right flank of each mouse.

Treatment of Animals

Treatment of animals started at day of randomization at a mean tumorvolume of ˜130 mm3, respectively. Mice are treated once weekly i.p. withthe different compounds as indicated in following table.

No of Route/Mode of animals Compound Dose (mg/kg) administration 10Xolair 10 i.p. once weekly 10 <VEGF> Avastin 10 i.p. once weekly 10<ANG-2> Ang2i- 10 i.p. once weekly LC06 10 XMAb1 10 i.p. once weekly

Monitoring of Tumor Growth

Animals were controlled 1× per week for their health status. Bodyweights were documented 1× per week after cell injection. The tumordimensions were measured by caliper on the staging day, at beginning oftreatment period once per week. Tumor volume was calculated according toNCI protocol (B. Teicher; Anticancer drug development guide, HumanaPress, 1997, Chapter 5, page 92) (Tumor weight =½ab², where “a” and arethe long and the short diameters of the tumor, respectively).

Termination criteria were the critical tumor mass (up to 1.7 g or Ø>1.5cm), body weight loss more than 20% from baseline, tumor ulceration orpoor general condition of the animals.

The results show that the bispecific bivalent <VEGF-ANG-2> antibodyXMAb1 showed a higher tumor growth inhibition in xenograft tumor modelColo205 in Scid beige mice compared to the treatment with monospecificantibodies (FIG. 12).

1. A bispecific, bivalent antibody comprising a first antigen-bindingsite that specifically binds to human VEGF and a second antigen-bindingsite that specifically binds to human ANG-2, wherein i) said firstantigen-binding site comprises as heavy chain variable domain (VH) theamino acid sequence of SEQ ID NO: 1, and as light chain variable domain(VL) the amino acid sequence of SEQ ID NO: 2; and wherein ii) saidsecond antigen-binding site comprises as heavy chain variable domain(VH) the amino acid sequence of SEQ ID NO: 3, and as light chainvariable domain (VL) the amino acid sequence of SEQ ID NO:
 4. 2. Thebispecific antibody according to claim 1 comprising a) a heavy chain anda light chain of a first full length antibody that specifically binds toVEGF; and b) a modified heavy chain and a modified light chain of a fulllength antibody that specifically binds to ANG-2, wherein the constantdomains CL and CH1 are replaced by each other.
 3. The bispecificantibody according to claim 2 wherein a) the heavy chain of the firstfull length antibody comprises the amino acid sequence of SEQ ID NO: 7,and the light chain of the first full length antibody comprises theamino acid sequence of SEQ ID NO: 5, and wherein b) the modified heavychain of the second full length antibody comprises the amino acidsequence of SEQ ID NO: 8, and the modified light chain of the secondfull length antibody comprises the amino acid sequence of SEQ ID NO: 6.4. The bispecific antibody according to claim 2 wherein a) the heavychain of the first full length antibody comprises the amino acidsequence of SEQ ID NO: 11, and the light chain of the first full lengthantibody comprises the amino acid sequence of SEQ ID NO: 9, and whereinb) the modified heavy chain of the second full length antibody comprisesthe amino acid sequence of SEQ ID NO: 12, and the modified light chainof the second full length antibody comprises the amino acid sequence ofSEQ ID NO:
 10. 5. The bispecific antibody according to claim 2, whereina) the heavy chain of the first full length antibody comprises the aminoacid sequence of SEQ ID NO: 15, and the light chain of the first fulllength antibody comprises the amino acid sequence of SEQ ID NO: 13, andwherein b) the modified heavy chain of the second full length antibodycomprises the amino acid sequence of SEQ ID NO: 16, and the modifiedlight chain of the second full length antibody comprises the amino acidsequence of SEQ ID NO:
 14. 6. A pharmaceutical composition comprisingthe antibody according to any one of claims 1 to
 5. 7. A method oftreating a patient suffering from cancer, comprising administering tothe patient an effective amount of the antibody according to any one ofclaims 1 to
 5. 8. The method of claim 7, wherein the cancer iscolorectal cancer, lung cancer, breast cancer, renal cancer, ovariancancer or glioblastoma.
 9. A method of treating a patient suffering fromvascular diseases, comprising administering to the patient an effectiveamount of the antibody according to any one of claims 1 to
 5. 10. Amethod of inhibiting angiogenesis in a patient having a pathologicalcondition associated with angiogenesis, comprising administering to thepatient an effective amount of the antibody according to any one ofclaims 1 to
 5. 11. An isolated nucleic acid encoding the bispecificantibody according to any one of claims 1 to
 5. 12. An expression vectorcomprising the nucleic acid according claim
 11. 13. A prokaryotic oreukaryotic host cell comprising the nucleic acid according to claim 11.14. A method for producing the bispecific antibody according to any oneof claims 1 to 5, comprising culturing the host cell of claim 13 so thatthe antibody is produced.
 15. The method of claim 14, further comprisingrecovering said antibody from the host cell.
 16. A bispecific antibodyobtained by the method of claim
 14. 17. A bispecific, bivalent antibodycomprising a first antigen-binding site that specifically binds to humanVEGF and a second antigen-binding site that specifically binds to humanANG-2, wherein the antibody comprises the amino acid sequences of SEQ IDNO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO:
 8. 18. A bispecific,bivalent antibody comprising a first antigen-binding site thatspecifically binds to human VEGF and a second antigen-binding site thatspecifically binds to human ANG-2, wherein the antibody comprises theamino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, andSEQ ID NO:
 12. 19. A bispecific, bivalent antibody comprising a firstantigen-binding site that specifically binds to human VEGF and a secondantigen-binding site that specifically binds to human ANG-2, wherein theantibody comprises the amino acid sequences of SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, and SEQ ID NO: 16.